摘要
肺炎克雷伯菌(Klebsiella Pneumoniae,K.pn)是革兰氏阴性菌,属于肠杆菌科克雷伯菌属,可在全身许多器官引发感染,但最高的发病机率在泌尿生殖道和肺部。当机体免疫力下降或者大量长期滥用抗生素引发菌群失调时导致感染,可引起脑膜炎、肺炎、泌尿系统发炎、伤口感染甚至败血症等,给临床治疗带来了许多麻烦,治疗不好则发病死亡率极高。虽然抗生素的使用,在目前可以有效治疗感染性疾病,可是当感染范围比较大,药物产生不良反应、多重耐药菌株逐年增加,而且停止使用后发生再次感染等因素,使抗生素治疗的临床使用遇到难题。由于人们对K.pn结构认识的加深,通过疫苗预防来防治K.pn侵袭增加了可行性,对于其疫苗的研究也逐渐升温,有重要的意义。
本研究首先对肺炎克雷伯菌的粘附素MrkD蛋白是否为主要保护性抗原进行了鉴定。采用原核表达得到纯化的粘附素MrkD蛋白,通过激光共聚焦显微镜观察粘附定位,进而通过粘附抑制与粘附动力学试验证明粘附素MrkD蛋白的粘附阻断作用,最后通过免疫保护实验,最终证实了菌毛粘附素MrkD蛋白可以作为肺炎克雷伯菌的主要保护性抗原,可用作进一步的表位疫苗研究。
为获取用于筛选粘附素MrkD蛋白B细胞表位的单克隆抗体,利用融合蛋白rMrkD作为免疫抗原,免疫BALB/c小鼠,利用淋巴细胞杂交瘤技术,采用间接ELISA筛选稳定分泌针对MrkD蛋白的单克隆抗体。经扩大培养后,制备单抗腹水,对所得单克隆抗体的效价、亲和力、特异性等进行鉴定。制备出了1株能稳定分泌抗MrkD蛋白的单克隆抗体杂交瘤细胞株E01,其中抗体亚类为IgG1,腹水效价为1:256,000,相对亲和力为0.3μg/mL,特异性反应显示,与大肠埃希菌,副伤寒沙门菌,铜绿假单胞菌,产单核细胞李斯特杆菌均无交叉反应性。为下一步筛选鉴定MrkD蛋白的B细胞表位提供基础。
为筛选鉴定MrkD蛋白的B细胞表位,用纯化的抗MrkD蛋白的单克隆抗体作为筛选配基,对噬菌体展示随机12肽文库进行三轮生物亲和淘选,高亲和力噬菌体得以富集;通过夹心ELISA、竞争ELISA鉴定出了33个阳性噬菌体克隆,为了找到核心序列,从中挑选16个阳性克隆进行序列测定和分析。发现阳性克隆大多呈递QKTLAKSTYMSA序列,其第3、4、5、9和11位置上的T、L、A、Y、S氨基酸与MrkD氨基酸残基(148-159位)有较高同源。说明T、L、A、Y、S可能决定了此B细胞表位的抗原性质,是该B细胞表位构成的关键氨基酸。使用带有QKTLAKSTYMSA序列的C1噬菌体克隆免疫BALB/c小鼠,通过ELISA和Western-blotting分析结果证实具有刺激小鼠产生抗体的能力,从而证明了M148-159是MrkD蛋白的B细胞表位。
对MrkD蛋白的Th细胞表位的筛选鉴定,Th细胞表位体外实验鉴定首先采用生物信息学软件预测MrkD蛋白可能的H-2d限制性Th表位,并合成表位肽,采用淋巴细胞增殖试验、流式细胞分析等实验方法进行了筛选和鉴定,获得BALB/c小鼠H-2d限制性Th细胞表位三条(M221-235,M175-189和M264-278),通过进行表位肽协同刺激试验,ELISA检测细胞因子试验,发现三个表位之间具有协同刺激效应,发现M221-235和M175-189为Th2细胞表位,M264-278为Th1细胞表位。Th细胞表位体内鉴定试验首先用已初步鉴定的表位多肽(M221-235,M175-189和M264-278)免疫BALB/c小鼠,进行淋巴细胞增殖试验来检测多肽免疫小鼠的细胞免疫应答,通过半定量RT-PCR检测多肽免疫小鼠脾淋巴细胞的细胞因子表达,发现三个表位肽能诱导机体产生特异性的CD+4 T淋巴细胞应答,表位肽致敏的淋巴细胞能识别相应多肽及rMrkD。
在筛选鉴定得到了1个B细胞表位和3个Th细胞表位的基础上,进行了肺炎克雷伯菌表位疫苗的设计、构建及免疫原性研究,通过三组柔性氨基酸将此四个表位依次串联并进行人工合成,构建重组质粒KMepi的。重组质粒转入大肠杆菌BL21,挑取单菌落以IPTG诱导表达,SDS-PAGE电泳观察蛋白表达情况,与对照组相比在31kD与45kD之间有目的蛋白的表达。纯化的EPI蛋白能诱导机体产生特异性细胞免疫应答,表位疫苗能诱导小鼠产生特异性抗体。
总之,本课题证实了MrkD蛋白作为主要保护性抗原,并筛选鉴定了MrkD的1个B细胞表位和3个Th表位,并对表位的免疫学特性进行了研究,在此基础上设计、构建了K.pn表位疫苗。构建的K.pn表位疫苗使包含的Th表位及B细胞表位各自发挥功能,为K.pn表位疫苗研究奠定了重要的实验基础。
Klebsiella pneumoniae is an important cause of respiratory infections and is responsible for significant morbidity and mortality in compromised individuals. Even though antibiotics are still the most effective treatment for K.pneumoniae infection, multiantibiotic resistance becomes a more pronounced problem.Thus, it is of great interest to find alternative ways to control the K.pneumoniae infection. Vaccination has become one of the most promising approaches against K.pneumoniae infection.
Currently, most of vaccines against K.pneumoniae were reported to use native components from K.pneumoniae such as fimbriae and capsular polysaccharide and LPS.In general, these treatment are based on a natural form of the pathogen. In response to K. pneumoniae infection, the host triggers vigorous humoral and cellular immune response.Though K. pneumoniae specific antibodies have been detected at high titers both in inflamed gastricmucosa and in serum, the infection is still persistent and even for life long, suggesting that K. pneumoniae can evade both adaptive and innate immune responses and the immune responses triggered by K. pneumoniae in nature were not optimal to eliminate this pathogen.The immune responses evoked by natural infection or subunit vaccine immunization are not favorable. The immune responses should be improved and modified.Therefore, we assume that the modified immunotherapy is a feasible treatment which may be effective to combat against K. pneumoniae. This purpose can probably be achieved by artificially promoting qualitatively or quantitatively immunity different from natural infection.
Epitope-based vaccines represent a modified immunotherapeutic approach which is based on the observation that in some instances. The potential advantages of the epitope-based approach include a specific immune response, increased safety, increased potency and breadth of rationally engineered epitopes and focusing on immune responses elicited by conserved epitopes.Accordingly, rational choices are made to isolate the components desired for the responses.The premise of these efforts is the identification of the appropriate epitopes. Phage display techniques have aroused interest as a new tool in the studies of mapping antigenic epitope since described by Smith.
It was demonstrated that MrkD adhesin mediated K.pneumoniae to adhere human respiratory tissue.It has not been investigated that the MrkD adhesin stimulate a protective immune response in vivo. In this study, we have investigated the ability of MrkD-specific antibodies to protect against infection using the murine model of acute K.pneumoniae infection.The ability of immune sera to passively protect animals from infection was determined. Based on these observations, MrkD adhesion was selected as a potential target for treatment.Mapping the epitope of MrkD adhesion is one of the essential steps for the development of antibodies against K. pneumoniae.
MrkD protein was expressed as a GST fusion protein in the E. coli BL21 to facilitate the purification.A pronounced band with the molecular weight of approximate 61 kDa was examined by SDS-PAGE in the supernatant of cell lysate after the induction,suggesting that the fusion protein was successfully expressed in the bacterial cells.MrkD—GST fusion protein was successfully purified by glutathione Sepharose 4B column chromatography and verified by SDS-PAGE.
Three hybridomas, E01—E03, were established by cell fusion.All mAbs were IgG1 and showed high specificity to MrkD protein.The titer of culture supernatant was 1:1 000—1:4 000 and the titer of ascites was 1:128,000—1: 1:256,000.The antigen were used in ELISA assay to calculate the affinity of the monoclonal anti-MrkD antibody using the method given in‘Materials and methods’section.MAb E01 displayed the highest affinity of about 0.3μg/mL. Competition studies using increasing concentrations of these three mAbs as inhibitors or competitors revealed that these three mAbs compete significantly with each other, indicating that recognition epitope of these three mAbs were the same or largely overlapped.
In order to map the epitope of MrkD antigen recognized by the mAb E01, the Ph.D.-12 library was screened with the purified mAb E01.After three rounds of biopanning, the selected phages bound to mAb were well enriched indicated by the increased recovery.Then 36 clones of the phages were randomly selected to test for their immunoreactivity with mAb E01.Among 36 clones, 33clones were positively recognized by mAb E01 while BSA was used as negative control.The DNA sequences of 16 positive clones were determined. There were eight different peptides in these 16 phage clones and most clones were the same QKTLAKSTYMSA sequence. Comparison between the protein sequence of MrkD and the dodecapeptide sequence revealed that amino acid sequences T, L, A, Y, S were conserved in MrkD(residues 148—159aa) and most positive phage clones.This result suggested that T, L, A, Y, S was the core amino acid of MrkD to form an epitope recognized by mAb E01.This notion was further confirmed by competitive inhibition ELISA.Pre-incubating mAb E01 with the recombinant phage clone C1 inhibited its binding with MrkD protein while pre-incubation with control phage, for example, phage clone C6 did not affect mAb’s binding with MrkD.
To evaluate the immune responses of the phage clones which comprised of T L A Y S amino acids, phage clone C1 and clone C6, were chosen to immunize the BALB/c mice using the intraperitoneal administration. The serum of the mice immunized with phage clone C1 showed higher binding with pre-coated MrkD on ELISA plate while the serum of the mice immunized with phage clone C6 showed the similar low binding with MrkD as controls as M13 phage.The MrkD proteins recognized by the antisera (raised by phage clones and the controls) were verified further by Western blot analysis.The antisera of clone C1 reacted specifically with the MrkD protein (61 kDa), while the pre-immune serum, the antisera induced by C6 clone and wild type M13 phage did not.
In order to study the immune response of BALB/c mice, the potential binding motifs for I-Ad and I-Ed in amino acid sequence of MrkD were scanned by the RANKPEP software. The I-Ad and I-Ed restricted epitopes were predicted, respectively. We selected five putative I-Ad restricted and one I-Ed restricted epitopes with higher scores. The selected peptides were synthesized and the purities of these peptides were all≥85% analyzed by high-pressure liquid chromatography.
Having identified three CD4+ T cell epitopes in MrkD protein, we then evaluated whether lymphocytes primed by peptides could recognize naturally processec antigen. For this purpose, we immunized mice with these peptides emulsified in IFA, and assessed the responses of CD4+ T cells in vitro, as described above. Meanwhile, we evaluated the responses of CD4+ T cells isolated from mice vaccinated with PBS in IFA as negative controls. T cells from BALB/c mice immunized with peptides M221-235, M175-189, and M264-278 exhibited significant proliferation upon in vitro stimulation with respective immunizing peptide and rMrkD. Under the same conditions, T cells from the PBS injected control mice did not respond to any peptide and rMrkD (SI < 2). Thus, these peptides can evoke the CD4+ T cell response and the CD4+ T cell induced by MrkD derived peptides react to rMrkD. The FACS analyses were also performed to determine the relative percentage of CD4+ CD3+ T cells immunized with peptides. The percentage of CD4+ CD3+ T cells from mice immunized with peptides M221-235, M175-189, and M264-278 were higher than the control group treated with PBS. These results reconfirmed that the peptides can stimulate the CD4+ T cells.
In conclusion, we have designed and constructed an epitope vaccine (KMepi) against K.pneumoniae. Although we have not assessed all the combination orders in the epitope vaccine, this study implies that epitope vaccine is a promising candidate for the development of K.pneumoniae treatment. Ongoing studies will evaluate the efficacies of other combination orders and compare the therapeutic effect of KMepi with rMrkD and K.pneumoniae lysates.
We will also further evaluate the therapeutic effect of the epitope vaccine in mice. The data from mouse models will provide much information for the further development of therapeutic vaccines against K.pneumoniae and other pathogenic microorganism, and should lead to studies of the epitope-based vaccine for human use.
引文
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