Human T cell recognition of the Mycobacterium leprae LSR antigen: epitopes and HLA restriction

We have in this work mapped epitopes and HLA molecules used in human T cell recognition of the Mycobacterium leprae LSR protein antigen. HLA typed healthy subjects immunized with heat killed M. leprae were used as donors to establish antigen reactive CD4+ T cell lines which were screened for proliferative responses against overlapping synthetic peptides covering the C-terminal part of the antigen sequence. By using this approach we were able to identify two epitope regions represented by peptide 2 (aa 29-40) and peptide 6 (aa 49-60), of which the former was mapped in detail by defining the N- and C-terminal amino acid positions necessary for T cell recognition of the core epitope. MHC restriction analysis showed that peptide 2 was presented to T cells by allogeneic cells coexpressing HLA-DR4 and DRw53 or DR7 and DRw53. In contrast, peptide 6 was presented to T cells only in the context of HLA-DR5 molecules. In conclusion, the M. leprae LSR protein antigen can be recognized by human T cells in the context of multiple HLA-DR molecules, of which none are reported to be associated with the susceptibility to develop leprosy. The results obtained are in support of using the LSR antigen in subunit vaccine design.     

结核分枝杆菌免疫优势抗原研究进展

结核病是当今影响人类健康、流行性最广、病死率最高的感染性疾病之一。结核病的诊断和疫苗的构建成为当前的研究热点,筛选出结核分枝杆菌免疫优势抗原是快速准确的诊断结核病及研制安全有效的疫苗的关键。拟对近年来国内外学者发现的结核分枝杆菌免疫优势抗原的分子生物学特性研究进展进行综述。     

Crystallization and preliminary X-ray crystallographic analysis of the 38-kDa immunodominant antigen of Mycobacterium tuberculosis.

The 38-kDa lipoprotein is one of the most potent cell surface immunogens of Mycobacterium tuberculosis in antibody-and T cell-mediated reactions. Using a pure recombinant form of the protein, we have recently shown that it binds phosphate much like that of the phosphate-binding protein (M(r) = 34.4 kDa) that is localized in the periplasm of Escherichia coli and is involved as an initial receptor for active transport of phosphate. The purified 38-kDa protein has been crystallized in 2 forms that are suitable for high-resolution structural analyses. One form belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 67.42 A, b = 113.38 A, c = 42.68 A, and beta = 108.53 degrees. The other is of orthorhombic space group P2(1)2(1)2 with a = 125.46 A, b = 72.27 A, and c = 73.43 A. Both crystal forms diffract to about 2 A resolution on a fine focus rotating anode.     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

结核分枝杆菌多表位诊断抗原的制备

将结核分枝杆菌(Mycobacterium tuberculosis,Mtb)多个B细胞预测表位串联表达(命名为B102),并初步评价其作为诊断抗原的血清学诊断价值。将MtbPstS1、ESAT6、CFP10、Ag85B、Ag85A及PPE54等6个蛋白的11个B细胞预测表位串联,加入合适的连接臂后全基因合成;将多表位片段插入带有TRX标签的表达质粒中,在大肠杆菌BL21(DE3)中诱导表达,并利用Ni~(2+)-Chelating亲和层析和DEAE阴离子交换层析纯化目的蛋白;利用Western blotting (WB)技术对目的蛋白抗原性进行鉴定,并建立Mtb抗体检测竞争法ELISA技术,初步评价此方法对阴阳血清样本的鉴别能力。目的蛋白以包涵体形式存在,其表达量约占菌体总蛋白的31.25%,经纯化及复性后蛋白B102可溶性存在,浓度为3.124mg/mL,纯度为96.71%;WB实验表明目的蛋白能与Mtb阳性血清相应抗体发生反应。对60份Mtb阳性血清及60份Mtb阴性血清进行检测得出其灵敏度为90.00%,特异性为93.33%,阳性预测值为93.10%,阴性预测值为90.32%,符合率为91.67%,McNemer检验的结果提示与"金标准"诊断结果无差异,Kappa=0.833,提示两种方法诊断结果一致性优异。原核表达与层析纯化可以获取抗原性优异的Mtb多表位诊断抗原,作为诊断抗原可以应用于Mtb的血清学检测中。     

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non‐synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species‐specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia‐related genes between M. bovis and M. tuberculosis.     

牛分支杆菌与肺结核分支杆菌基因组的比较

通过比较基因组学的方法研究发现,牛分支杆菌与肺结核杆菌基因组的同源性为99．95％,但在牛分枝杆菌基因组中有11个缺失区,大小从1kb到12．7kb,遗传信息的缺失引起牛分枝杆菌的基因组减小;牛分枝杆菌与肺结核分枝杆菌H37Rv间存在着2437个单核苷酸多态性（SNPs）,与肺结核分枝杆菌CDC1551间存在着2423个单核苷酸多态性（SNPs）,牛分支杆菌与肺结核分枝杆菌在编码细胞壁和分泌蛋白上变异程度也是巨大的。研究结果揭示了牛分支杆菌与肺结核分枝杆菌的遗传关系,为研究分支杆菌疫苗和诊断试剂提供理论依据,对牛肺结核病的防治有着非常重要的意义。     

Identification of the new T-cell-stimulating antigens from Mycobacterium tuberculosis culture filtrate

The proteins secreted by Mycobacterium tuberculosis are an important target for vaccine development. To identify the antigens from M. tuberculosis culture filtrate (CF) that strongly stimulate T-cells, the CF was fractionated by ion-exchange chromatography and then non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mini-whole gel elution. Each fraction was screened for its ability to induce interferon-gamma (IFN-gamma) production in peripheral blood mononuclear cells isolated from healthy tuberculin reactors. The protein bands that strongly induced IFN-gamma production were subjected to N-terminal sequencing. Two new proteins, a 17-kDa protein (Rv0164, MTSP17) and an 11-kDa (Rv3204, MTSP11) protein, were identified. The recombinant MTSP17 (rMTSP17) and rMTSP11 induced significant production of IFN-gamma and interleukin (IL)-12p40 in peripheral blood mononuclear cells from healthy tuberculin reactors. Interestingly, IL-12p40 production in response to rMTSP11 was significantly higher than that in response to rMTSP17 or the three components of the antigen 85 complex. These results suggest that MTSP11 antigen should be further evaluated as a component of a subunit vaccine.     

结核分枝杆菌与巨噬细胞相互作用的研究进展

结核分枝杆菌（Mycobacterium tuberculosis,MTB）是一种典型的胞内致病菌,巨噬细胞是MTB在体内的主要宿主细胞。巨噬细胞具有强大的吞噬功能,在机体固有免疫和适应性免疫中均发挥着重要作用,可有效保护宿主免受结核分枝杆菌的感染。MTB在与宿主巨噬细胞的长期相互作用过程中,逐渐形成多种逃避杀灭的有效策略,得以在宿主体内存活并增殖。该文从巨噬细胞抗MTB感染及MTB逃避巨噬细胞杀灭两个方面综述国内外的研究进展。     

Profile of a 24 kDa Mycobacterium tuberculosis antigen in the urine samples of tuberculosis patients under therapy regime

Secretion of a low molecular weight (24 kDa) Mycobacterium tuberculosis-specific antigen was analysed in the urine samples of tuberculosis patients under antimycobacterial therapy regime. The urine samples of sputum-positive and culture-positive tuberculosis patients under therapy regime were collected after 2, 3, 4, 5 and 6 months of antimycobacterial therapy. After concentration of the samples by ultrafiltration the proteins were resolved by SDS–PAGE. The antibodies raised in rabbits against M. tuberculosis strain H₃₇Ra were used to check specificity of the bands by Western blotting. It was found that the tuberculosis patients secreted a 24 kDa M. tuberculosis-specific antigen in their urine. This band was present in the samples taken upto 5 months of drug therapy but was absent in the samples taken after 6 months of antimycobacterial therapy. This study gives evidence in support of the continuation of chemotherapy of tuberculosis patients for at least 6 months. Also, the 24 kDa excreted/secreted antigen can be used as a marker for monitoring the drug therapy as well as for the diagnosis of tuberculosis patients. Moreover, the urine sample taken in the study is a non-invasive and safer sampling method as compared to sampling blood or sputum which is the usual procedure in these patients.     

结核分枝杆菌可视化抗体检测蛋白芯片的制备

目的：利用克隆表达的7种结核分枝杆菌优势表位抗原,建立可视化抗体检测蛋白芯片,用于结核病辅助诊断。方法：将7种结核分枝杆菌优势表位抗原,即38kD、ESAT-6、CFP10、MPT64、Mtb8、Mtb8.4和Mtb16.3点于修饰的基片上,制备可检测7种结核抗体的多靶点蛋白微阵列,建立免疫金银染色检测系统;使用该芯片对48例临床结核病患者血液样品进行检测,并与“金标准”痰涂片（48例）和痰培养（其中的29例）检测结果进行比较,分析其敏感性;对30名献血员血液样品进行检测,分析其特异性。结果：可视化抗体检测蛋白芯片的敏感性分别为98.5％和96.6％,而痰涂片和痰培养检测方法的敏感性分别为35.4％和48.3％;可视化抗体检测蛋白芯片的特异性为93.3％。结论：建立的结核分枝杆菌可视化抗体检测蛋白芯片检测敏感性显著高于痰涂片和痰培养方法,可用于结核病的临床辅助诊断,提高痰涂片和痰培养假阴性的检出率。     

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0-96·0%) and specificity (98%, 95% CI: 89·0-100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.     

结核杆菌抗原85A与小鼠白细胞介素21共表达DNA疫苗的构建及其免疫效应研究

目的:利用真核表达质粒pRSC,构建结核杆菌抗原85A(Ag85A)与小鼠白细胞介素21(mIL21)共表达重组体pRSC-mIL21-Ag85A,为研究新型结核杆菌DNA疫苗提供新的策略。方法:从质粒pcDNA3.1-mIL21中经PCR扩增出mIL21基因,并插入质粒pRSC中构成pRSC-mIL21;再从pIRES-Ag85A质粒中经PCR扩增出Ag85A基因,构建于pRSC-mIL21重组质粒上,成为共表达DNA疫苗pRSC-mIL21-Ag85A。结果:经酶切、基因测序证实,该疫苗构建正确并能成功表达目的基因。共表达DNA疫苗免疫小鼠后,CTL活性、特异性淋巴细胞增殖水平及小鼠血清特异性抗体均呈有意义的提高。结论:结核杆菌Ag85A与mIL21共表达DNA疫苗能诱导小鼠免疫反应,为进一步研究DNA疫苗抗结核杆菌攻击的免疫防护效应奠定了基础。     

Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis

The gene encoding a major protein antigen of Mycobacterium tuberculosis has been cloned and sequenced using oligonucleotide probes derived from the N-terminal sequence of the analogous protein from Mycobacterium bovis BCG. The gene analysis revealed a sequence encoding a protein of 99 amino acid residues, with a molecular mass of 10.7 kDa. Computer prediction suggests that the protein contains three T-cell-determined epitopes (of which one has been demonstrated experimentally) and three B-cell-determined epitopes. The protein sequence was homologous to two prokaryote heat-shock proteins and the gene possessed heat-shock-like promoter sequences upstream of the initiation codon. A hairpin loop identified in the upstream sequence may also be important in regulation of the gene.     

PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a novel immunodominant antigen of Mycobacterium tuberculosis induces humoral and cellular immune responses in mice

Subtractive DNA hybridization of pathogenic M. bovis and BCG, and comparative genome-wide DNA microarray analysis of M. tuberculosis H37Rv and BCG identified several RD, designated as RD1 to RD16, between M. tuberculosis and M. bovis on the one hand and BCG on the other. These regions cover 108 ORF of M. tuberculosis H37Rv, and are deleted from all 13 BCG sub-strains currently used as anti-tuberculosis vaccines in different parts of the world. In this study, we evaluated cellular and humoral immune response in C57BL/6 mice immunized with the PPE protein Rv3425, encoded by an ORF found in RD11 of M. tuberculosis. Rv3425 protein induced an increased Th1/Th2 type immune response in mice, characterized by an elevated concentration of IFN-gamma in antigen stimulated splenocyte culture and a strong IgG(1) antibody response. These results provide evidence on the immunogenicity of the PPE protein Rv3425 which, together with its reported immunodominant characteristics, imply that it may be a candidate for development of a vaccine for the control of TB.     

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.     

结核分枝杆菌furA基因真核表达质粒的构建及表达

以BamH Ⅰ和Hind Ⅲ双酶切pRSET-furA,获得结核分枝杆苗铁调控基因furA,将其克隆入真核表达载体pcDNA3．1（-）,构建了重组质粒pcDNA-furA,经酶切鉴定正确后,将重组质粒以阳离子聚合物转染CHO细胞。经RT—PCR分析表明,furA可在CHO细胞中转录;用间接免疫荧光检测,表达有FurA蛋白的细胞着染。以上结果表明,通过构建结核分枝杆菌furA基因的真核表达载体pcDNA-furA,使知以基因可以在CHO细胞中表达。     

结核分枝杆菌融合抗原Rv3914-6His的原核表达、纯化与抗原性检测

血清学诊断是目前临床诊断中应用最为广泛的方法之一,用原核表达并鉴定了系列结核分枝杆菌抗原融合蛋白Rv1908c-6His、Rv0733-6His、Rv0899-6His、Rv1411c-6His和Rv3914-6His,并以此包被酶标板,以Rv2031c-6His为阳性对照,采用间接ELISA方法比较了34例活动性结核病患者与35例健康体检者血清中抗结核分枝杆菌抗原的IgG水平。结果显示,活动性结核病患者中针对Rv1411c-6His、Rv3914-6His和Rv2031c-6His的IgG水平明显高于健康体检者,其中Rv3914-6His的AUC值(0.786 7)略高于目前临床应用的类似于16 ku的抗原Rv2031c-6His(AUCRv2031c=0.754),提示Rv3914抗原可能是结核病血清诊断的新的候选标志物。