【作者】 张建辉；

【导师】 陈建平；

【作者基本信息】 四川大学 ， 基础医学， 2023， 博士

【摘要】 内脏利什曼病是由杜氏利什曼原虫和婴儿利什曼原虫通过白蛉的虫媒传播而感染人和狗。内脏利什曼病的临床症有不规则发热、肝脾肿大和贫血,若得不到及时的治疗将因为肝功能和免疫系统的耗尽危及生命。该病曾在我国一度流行,并在上世纪60-90年代通过公共卫生干预措施基本上消除内脏利什曼病。如今个别省份依旧零星出现这种病例,而且在第三世界国家依旧流行。目前用于治疗该病的药物有五价锑剂,两性霉素和米替福星,由于药物的毒副作用和价格不菲,因此疫苗是应对该病的理想策略。世界上还没有正式用于人身上的疫苗,内脏利什曼病疫苗的研究仍有着很长的路要走。本课题通过细胞感染实验和前期动物实验确认了两株具有毒力差异的杜氏利什曼原虫,即9044和DD8株。两虫株的毒力差异可能是由于它们的表达蛋白不同导致的,即差异表达蛋白。在这些差异表达蛋白里,有些蛋白可能是上调表达的毒力因子,有的是为了免疫逃逸而下调的抗原蛋白。毒力因子和抗原蛋白都适合充当疫苗研究里的候选疫苗。如今的疫苗研究结合了多学科,从不同的角度深入揭示抗原蛋白的保护性,提供了更为科学和创新的研究方法。本课题通过建立两虫株的差异蛋白质组学,在这些差异表达蛋白基础上进行表位的预测,筛选出适合充当抗原的差异表达蛋白。如果差异表达蛋白的毒力因子和抗原只要它们适合充当抗原蛋白,那它们将具有一定数量的表位被预测和筛选出来。抵抗内脏利什曼病的有效免疫反应是Th1免疫反应,Th1免疫反应的诱导在于树突细胞分泌的IL-12。沃尔巴克氏菌表面蛋白(Wolbachia surface protein,WSP)已被报道可以激活Toll样受体2(Toll-like receptor 2,TLR2)和Toll样受体4(Toll-like receptor 4,TLR4),进而促进抗原提呈细胞的成熟和产生相关的细胞因子,如IL-12、TNF-α和IL-10等。所以WSP作为拟佐剂可能促进抗原提呈和引导Th1免疫反应。先前的研究报道这种蛋白作为拟佐剂可能适用于利什曼原虫的疫苗。由于还未有被公认适合用于抗利什曼原虫疫苗的佐剂,并且WSP还没真正和疫苗一起应用,因此本课题表达出重组沃尔巴克氏菌表面蛋白(recombinant Wolbachia surface protein,rWSP),验证了它刺激树突细胞和巨噬细胞TLR2和TLR4后的效应。这些效应包括信号通路下游关键分子的磷酸化上调、相关效应分子m RNA转录和生成的提高,并确认rWSP能够激活抗原提呈细胞。将rWSP作为拟佐剂和筛选出来的抗原蛋白一起进行动物的免疫攻毒实验以验证此次筛选的抗原蛋白的疫苗效应和rWSP的佐剂效果。综合差异表达蛋白的生物信息学分析、重组蛋白的表达、rWSP刺激抗原提呈细胞的研究和动物免疫攻毒实验,本课题分成四个部分:第一部分,提取杜氏利什曼原虫强弱毒株(9044株和DD8株)的总蛋白,用质谱检测以建立差异表达蛋白质组学;应用Vaxijen、Alg Pred和TBtool对9044株和DD8株的差异表达蛋白进行抗原指数、变应原性、人和小鼠蛋白同源性的预测以进行第一筛选;应用免疫生物信息学将第一次筛选出的差异表达蛋白和临床实验阶段的利什曼原虫疫苗利什曼原虫延伸启始因子(Leishmania elongation initiation factor,Le IF)、巯基特异性抗氧化蛋白(Thiol-specific antioxidant protein,TSA)、硕大利什曼原虫真核应激诱导蛋白1(Leishmania major homologue to eukaryotic stress-inducible protein 1,Lm STI1)、和Leish-111f(由Le IF、TSA和Lm STI1组成)进行人的MHCⅠ、MHCⅡ和IFN-γ表位的预测并计数各种表位丰富度以进行第二次筛选;应用C-Imm Sim server将第二次筛选结果进行计算机免疫模拟,计算IFN-γ与IL-10比值,并结合IFN-γ、MHCⅠ和Ⅱ的表位丰富度进行聚类以进行第三次筛选;将第三次筛选的结果进行结构域的预测、表位聚集的可视化、三维结构的建模和模型评估,并选出抗原蛋白。第二部分,用PCR扩增出抗原蛋白和rWSP的目的基因蛋白质编码区(Coding sequence,CDS)序列,然后胶回收目的片段并通过双酶切和T4连接酶的方法将目的片段插入p ET30a的多克隆位点中。将重组的p ET30a转化E.coli DH5α中以克隆重组质粒,并通过菌落PCR和测序来鉴定质粒。将正确的重组质粒转化E.coli BL21中进行诱导表达。随后通过超声裂解获得表达产物并通过SDS-PAGE电泳和Western Blot鉴定表达产物的正确性。最后对表达产物进行纯化和复性透析,为后续实验提供所需的重组蛋白。第三部分,用GM-CSF因子诱导骨髓细胞分化为树突细胞,提取腹腔单个核细胞培养为巨噬细胞。将rWSP分别刺激树突细胞和巨噬细胞,在相应的时间点提取细胞的总蛋白、m RNA和细胞上清液。应用Western Blot探索总蛋白中TLR2和TLR4的信号通路下游关键分子p65蛋白(p65)、p38蛋白(p38)、胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK)、c-Jun氨基末端激酶(c-Jun N-terminal kinases,JNK)和磷酸化蛋白激酶B(protein kinase B,PKB)(又称Akt)的磷酸化程度。应用q PCR探索树突细胞中IL-12、TNF-α、IL-10和巨噬细胞诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)分子的m RNA转录情况。用ELISA和Griess法分别检测树突细胞上清中IL-12、TNF-α、IL-10和巨噬细胞NO的生成情况。第四部分,将抗原蛋白分别和铝剂进行乳化,再分别皮下免疫BALB/c小鼠,随后进行腹腔感染杜氏利什曼原虫9044株。在感染后6周通过检测血清的抗体、肝脏的病理情况和脾脏的载虫量来评估抗原蛋白的免疫保护性以确定候选疫苗。确定的候选疫苗混为一起作为Mix,结合rWSP进行皮下免疫和9044株腹腔感染BALB/c小鼠,在特定的时间检测血清的抗体,抗原刺激脾脏细胞的细胞因子、T细胞比例、肝脏病理情况和脾脏的载虫量以评估候选疫苗Mix结合rWSP的免疫保护效果。结果表明,在强弱毒株的差异蛋白质组学基础上,经过表位预测、表位丰富度和计算机免疫模拟等生物信息学分析,筛选出三种抗原蛋白:氨基酸通透酶(Amino acid permease(60-450 AA))、Amastin类似蛋白(Amastin-like(全部序列))和假设蛋白(Hypothetical protein(XP＿003865405.1)(20-300 AA))。通过大肠杆菌成功表达三种抗原蛋白和rWSP并纯化。rWSP刺激树突细胞和巨噬细胞后,在相应时间点的p65、p38、ERK、JNK和Akt发生了很大程度的磷酸化,树突细胞的IL-12、TNF-α、IL-10和巨噬细胞的i NOS的m RNA转录很大程度的提高,以及树突细胞的IL-12、TNF-α、IL-10和巨噬细胞的NO具有很高的生成水平。TLR2和TLR4的下游信号分子磷酸化表明树突细胞和巨噬细胞被活化,树突细胞产生TNF-α和IL-12以及巨噬细胞产生NO说明rWSP可能引导Th1的免疫反应和促进抗原提呈。rWSP的细胞效应研究和先前研究报道结果相符。抗原蛋白Amino acid permease、Amastin-like和Hypothetical protein都使得小鼠产生高滴度特异性的抗体,促使肝脏的炎症细胞灶形成,减少脾脏的无鞭毛体数量,其中Amastin-like减虫率61%,效果最好。将这三种抗原蛋白作为候选疫苗,并混成Mix。相比较于Infected control和Normal control组,Mix和Mix+rWSP组具有以下免疫效应:产生高滴度的抗体,较小的Ig G1/Ig G2a比值;脾脏细胞的抗原刺激后产生较高水平的IFN-γ、TNF-α、IL-12、IL-4和IL-10,具有较大的IFN-γ/IL-4比值和CD4~+和CD8~+T细胞比例;肝脏产生较多的炎症细胞灶,以及脾脏的载虫量是最少的。其中,Mix+rWSP组产生的免疫效果一定程度上优于Mix组(感染第6和12周,Mix减虫率分别是65.8%和84.5%,Mix+rWSP减虫率分别是80.6%和88.7%)。本课题创新之处在于:1.创建了基于差异蛋白质组学和生物信息学的新抗原蛋白筛选体系;2.探索了Amino acid permease、Amastin-like和Hypothetical protein和它们的Mix具有一定免疫原性和保护性;3.证实了rWSP刺激树突细胞和巨噬细胞的TLR2和TLR 4效应和结合疫苗在动物实验上的佐剂效果。总之,本课题筛选新抗原蛋白的方法、发现的新抗原和拟佐剂的研究为抗利什曼原虫疫苗和其他的疫苗研究提供了新思路。更多还原

【Abstract】 Visceral leishmaniasis(VL)caused by Leishmania donovani(L.donovani)and Leishmania infantum(L.infantum)is transmitted to human and canine through the vector borne transmission of sandfly.VL is characterized with irregular fever,hepatosplenomegaly and anemia.If left untreated,VL is the most fatal because of the exhaustion of hepatic and immune function.The disease was once endemic in our country and essentially eliminated through public health interventions in the 1960s-1990 s.Sporadic cases occur in some provinces today and are prevalent in third world countries.The drugs currently employed for VL treatment are pentavalent antimony agents,amphotericin and mitofusin.These drugs bring side effects and high cost,and vaccine application is a desirable strategy.None of vaccine against VL has been used clinically and there is still a long way to go.We have determined the virulent differences between L.donovani 9044 and DD8 through cellular experiments and previous animal experiments,and differentially expressed proteins may be as the results of virulent differences.Among these differentially expressed proteins,some may be up-regulated virulent factors or downregulated antigenic proteins for immune escape,which of them are suitable to serve as vaccine candidates in vaccine researches.Nowadays,vaccine studies combine multidisciplinary to further reveal the antigenic properties from various perspectives,providing more scientific and innovative approaches for vaccine researches.To select the desirable antigenic proteins,epitope was screened in silico on the basis of differentially expressed proteins between 9044 and DD8 strains that had been established by LC-MS/MS.Virulence factors and antigens down-regulated for immune escape are appropriate for vaccine candidates and they will meet the rules of epitope prediction.Most vaccines against VL should establish Th1 response that induced by IL-12 derived from DC cells.Wolbachia surface protein(WSP)have been reported to activate Toll-like receptor 2(TLR2)and Toll-like receptor 4(TLR4)to promote the maturation of antigen-presenting cells and the production of associated cytokines such as IL-12,TNF-α and IL-10,etc.Therefore,WSP as a adjuvant might promote antigen presentation and induce Th1 immune responses.It was reported in previous studies that WSP might be suitable for the role of adjuvant in the studies of vaccines against VL.None of suitable adjuvant against Leishmania is confirmed by public and WSP has not really been used with a vaccine.Therefore,we expressed recombinant WSP(rWSP)to discover its properties.When rWSP was cultured with DC cells and macrophages,the phosphorylation of key downstream molecules of the Toll receptor2 and 4 signaling pathways,m RNA transcription,cytokines and NO expression were found up-regulated in cells to activate antigen-presenting cells.The selected antigenic proteins as vaccines and rWSP as adjuvant were combined to study the respective efficacy of vaccine and adjuvant through animal experiments.According to immunobioinformatics,recombinant protein expression,the studies of rWSP acting on DC cells and macrophages and animal experiments,four parts of our studies are represented as follow:In the first part,differentially expressed proteins between virulent(9044)and avirulent(DD8)strains were generated by using liquid chromatography-mass spectrometry/mass spectrometry that analyzed the total proteins of 9044 and DD8 strains.In the first step,analyses,including antigenic index,allergenicity,and the similarity of humans and mice,were performed respectively by Vaxijen,Alg Pred and TBtool software.In the second step,the differentially expressed proteins filtered in the1 st step and four vaccines against VL in clinical trials Leishmania elongation initiation factor(Le IF),Thiol-specific antioxidant protein(TSA),Leishmania major homologue to eukaryotic stress-inducible protein 1(Lm STI1),and Leish-111f(composed with Le IF,TSA,Lm STI1)were further predicted their MHC Ⅰ,MHC Ⅱ and IFN-γ epitopes using immunobioinformatics,and the percentages of epitope peptide were also analyzed.In the third step,the differentially expressed proteins filtered in the second step were subjected to C-Imm Sim server to analyze their ratio of IFN-γ to IL-10.The value of IFN-γ/L-10 along with the percentage of MHC Ⅰ,MHC Ⅱ and IFN-γ epitope peptide were used to cluster differentially expressed proteins by heat map to identify desirable antigenic proteins.The results of the 3rd step were subjected to domain prediction,epitope aggregation visualization,3D structure modeling and model evaluation to screen antigenic proteins.In the second part,the coding sequences of antigenic proteins and rWSP were amplificated by PCR and purified using Universal DNA Purification Kit.These PCR productions were cloned into p ET30 a by double digests and T4 connection.Recombinant p ET30 a plasmids were transferred into E.coli DH5α for amplification,which were identified by clone PCR and sequencing.E.coli BL21 transferred with recombinant p ET30 a were induced to express proteins and lysed by ultrasonic disruption.Cellular lysate was confirmed with SDS-PAGE and Western Blot.Recombinant proteins were purified and renatured for the proteins required by followup experiments.In the third part,the bone marrow cells were induced to differentiate into dendritic cells with GM-CSF factor,and peritoneal mononuclear cells were extracted and cultured as macrophages.Dendritic cells and macrophages were cultured with rWSP,and total protein,m RNA and cell supernatant were extracted from the cells at the corresponding time points.Western blot was applied to explore the phosphorylation of downstream key molecules of Toll-like receptor 2 and 4 signaling pathway including p65 protein(p65),p38 protein(p38),extracellular signal-regulated kinase(ERK),cJun N-terminal kinases(JNK)and protein kinase B(known as Akt)of in total protein.The transcription of IL-12,TNF-α and IL-10 in DC cells and inducible nitric oxide synthase(i NOS)in macrophages were analyzed by q PCR.IL-12 TNF-α and IL-10 from DC cell supernatant were tested by ELISA and NO from macrophage supernatant was tested by Griess.In the fourth part,BALB/c mice were immunized subcutaneously with antigenic proteins along with alum respectively and challenged intraperitoneally with L.donovani 9044 strains.The immunoprotection of antigenic proteins were assessed at week 6 after infection by detecting antibodies in serum,hepatic pathology,and splenic parasite burdens to identify vaccine candidates.The identified vaccine candidates were mixed together as mix and combined with rWSP for immunization.L.donovani 9044 intraperitoneally infected BALB/c mice.Antibodies in serum,cytokines and T cell proportion of splenocytes cultured with vaccine mix,hepatic pathology,and splenic parasite burdens at specific times were analyzed to evaluate the immunoprotection of the vaccine candidate mix combined with rWSP.The results showed that on the basis of differential proteomics between virulent and avirulent strains,Amino acid permease(60-450 AA),Amastin-like(all)and Hypothetical protein(XP＿003865405.1)(20-300 AA)were screened as antigenic proteins through the bioinformatics analysis of epitope prediction,epitope abundance and immune simulation in silico.Antigenic proteins and rWSP were successfully expressed by E.coli and purified for the proteins required by follow-up experiments.After stimulation of dendritic cells and macrophages with rWSP,p65,p38,ERK,JNK,and Akt were phosphorylated to a significant extent at corresponding time points.IL-12,TNF-α and IL-10 transcription from DC cells and i NOS transcription from macrophages were largely elevated.The levels of IL-12,TNF-α and IL-10 expression from DC cells and NO expression from macrophages were also improved.In a word,the phosphorylation of downstream key molecules from TLR2 and TLR4 signaling pathway indicated the activities of DC and macrophage,and the TNF-α and IL-12 from DC and NO produced by macrophage suggested rWSP may promote Th1 response and antigen presention.rWSP can stimulate antigen-presenting cells and produce effector molecules,which converges with the results of previous studies.Amino acid permease,Amastin-like,and Hypothetical protein all produced high titer specific antibodies,promoted foci of inflammatory cells in the liver and reduce the number of amastigotes in the spleen.To sum up,these three antigenic proteins are immunogenic and protective,with amastin-like(reduction rate: 61%)having the best efficacy.Amino acid permease,amastin like,and hypothetical protein are selected as vaccine candidates and mixed as vaccine candidate mix.Comparing with the results from infected control and normal control groups,Mix and Mix + rWSP groups performed well in some aspects as follow: 1.higher titers of antibody along with small Ig G1/Ig G2 a ratio were produced.2.stimulating splenocytes with vaccine candidate mix led to higher levels of IFN-γ,TNF-α,IL-12,IL-4 and IL-10 production,larger IFN-γ/IL-4 ratio and CD4 and CD8 proportion.Mix + rWSP group produced more hepatic foci of inflammatory cells and had 80.6%(six weeks after infection)and 88.7%(twelve weeks after infection)reduction rates fewer Leishmania in spleens than Mix group(65.8% and 84.5% reduction rates at sixth and twelfth week after infection,respectively).The innovation of our studies includes the following aspects: 1.A new antigen protein screening system based on differential proteomics and bioinformatics was established.2.That Amino acid permease,Amastin-like and Hypothetical protein and their Mix have certain immunogenicity and protection was found.3.The TLR2 and TLR4 effects of dendritic cells and macrophages stimulated with rWSP and the adjuvant effects of rWSP in animal experiments were confirmed.In conclusion,our screening neoantigen protein methods,novel vaccine efficacy and the studies of potential adjuvants provide new ideas for anti-Leishmania and other vaccine researches.更多还原