HL-60分化细胞表面标志、活性及其对肺炎链球菌调理吞噬杀菌能力的动态变化

目的分析HL-60分化细胞的表面标志、活性及其对肺炎球菌调理吞噬杀菌能力的动态变化。方法以流式细胞仪连续监测分化1～7 d的HL-60细胞表面标志CD11b、CD35和CD71的表达以及活细胞、凋亡细胞和死亡细胞的比例,同时用09CS、QC2、B、C和F 5份质控血清以调理吞噬杀菌试验检测肺炎链球菌血清型6B、7F、14和23F的杀菌滴度。结果分化3～6 d的HL-60细胞表面标志、活细胞比例可达到实验室要求,5份质控血清的调理吞噬杀菌滴度稳定而且在质控范围之内。结论分化3～6 d的HL-60细胞可以用于评价肺炎链球菌疫苗免疫血清的调理吞噬杀菌试验,为调理吞噬杀菌试验的建立和标准化提供了依据。     

不同品牌胎牛血清用于肺炎链球菌调理吞噬作用的比较

目的 比较不同品牌的胎牛血清用于HL-60细胞分化,以及配制成调理缓冲液后对肺炎链球菌调理吞噬杀菌力的影响。方法 采用5个不同品牌(品牌1~5)的胎牛血清用于HL-60细胞分化培养液和调理缓冲液的配制,计算分化后细胞活力,并进行肺炎链球菌调理吞噬杀菌试验,比较对照孔菌数和质控血清的调理指数。结果 与品牌1胎牛血清分化细胞的活力相比较,品牌5分化的细胞活力差异具有统计学意义(P=0.023,P<0.05),而品牌2、3、4分化细胞的活力差异均不具有统计学意义(P=0.129、0.186、0.440,P>0.05);使用品牌5分化的细胞,对3、6A、7F、9V、14、18C、19A、19F血清群/型调理指数因为调理吞噬后菌落数过少,无法计算。使用品牌5配制的调理缓冲液,对3、9V、19F血清群/型调理指数因为调理吞噬后菌落数过少,无法计算。结论 为保障肺炎链球菌调理吞噬试验的稳定性,建议对试验过程中使用的胎牛血清进行筛选。     

多型调理吞噬试验方法的长期稳定性

目的通过对2010—2016年间09CS针对13个型别的肺炎链球菌荚膜多糖特异性抗体滴度的分析,探讨调理吞噬杀菌试验(multiplexed opsonophagocytic killing assays,MOPA)的长期稳定性。方法统计2010—2016年检测人肺炎链球菌质量控制血清(质控血清)09CS中针对1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、23F共13个血清型的OPKA滴度,分别计算2010、2011、2012、2013、2014—2016年09CS针对各个型别的OPKA滴度的GMT值及其95%可置信区间;统计09CS针对13个血清型的OPKA滴度在质量控制范围之内的比率;计算2010—2016年09CS针对13个血清型各型别的OPKA滴度CV值。结果从OPKA滴度来看,2010年普遍较低,而2011年、2012年、2013年、2014—2016年这几年间的09CS针对13个血清型的滴度结果稳定。2011—2016年期间检测结果在质控血清滴度范围的比率:2010年最低值是4%,均值为30%;2011年最低值是65%,均值为80%;2012年最低值是73%,均值为85%;而2013年最低值为77%,均值为94%;2014—2016年最低值为71%,均值为90%。2010—2016年09CS针对13个血清型的OPKA滴度各型别CV:各年份的平均值分别为:125%、64%、47%、41%和39%。结论充分证明了从2011年起本实验室建立的MOPA稳定性良好。     

质控血清09CS中13个肺炎球菌荚膜多糖抗体IgG含量检测范围的确定

目的 建立09CS作为质控血清用于13价肺炎球菌多糖蛋白结合疫苗(13PCV)临床血清样本检测中的检测值范围。方法 用WHO推荐的检测人血清中抗肺炎球菌荚膜多糖抗体IgG的定量ELISA,以国际人肺炎球菌标准血清007sp为标准,将09CS作为待测血清,检测其在13个血清型(1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、23F型)中抗荚膜多糖抗体IgG含量的值。连续检测09CS血清100余次,计算99%置信区间的各血清型几何平均抗体浓度、标准偏差(SD)和变异系数(CV)。结果 检测得到09C中13个血清型抗荚膜多糖IgG抗体含量以及在99%置信区间(CI)下±2.58倍SD的检测值范围;13个血清型检测结果的CV分别为10.86%、12.52%、13.96%、14.98%、28.77%、11.16%、14.96%、9.31%、10.43%、7.28%、10.86%、12.52%、13.96%,除6A型外,各型CV均低于15%,表明试验间精密度良好;检测次数异常率低于10%。结论 09CS可作为质控血清,用于13价肺炎球菌结合疫苗临床血清中抗荚膜多糖抗体IgG含量的ELISA检测。     

11价肺炎链球菌多糖-蛋白D结合疫苗在婴儿中的免疫原性和安全性[英]Narkka A…／／Pediatr Infect Dis J．

在过去的10年中，对于一系列以不同蛋白和多糖作为载体的多糖．蛋白复合物型抗肺炎链球菌疫苗的研究表明，在众多的疫苗中，只有1种带有CRM197载体蛋白的7价疫苗(PncCRM)能够有效地阻止致病性肺炎链球菌的感染。在欧美地区，这种疫苗广泛应用于高危人群，但其血清型是4、6B、9V、14、18C、19F和23F，缺少了亚洲等众多发展中国家普遍存在的血清型1和5。为了有效地抑制更多型别的肺炎链球菌，     

人肺炎球菌参考血清09CS中11个血清型抗荚膜多糖的抗体IgG含量的定值

对人肺炎球菌参考血清09CS中11个肺炎球菌血清型(2、8、9N、10A、11A、12F、15B、17F、20、22F、33F)的抗荚膜多糖抗体IgG含量进行定值。方法用WHO推荐的标准检测人血清中抗肺炎球菌荚膜多糖抗体IgG定量ELISA方法,以国际标准血清89SF为标准,对此11个血清型抗荚膜多糖抗体IgG含量进行定值;以暂定的09CS的定值为标准检测12份WHO校正血清、16份兰州生物制品研究所有限责任公司(LIBP)质控血清和89SF,对定值的准确性进一步验证。结果以09CS的定值为标准检测的12份WHO校正血清和16份LIBP质控血清的11个血清型抗荚膜多糖抗体结果,与以89SF为标准检测的结果,均具有良好的直线相关关系(r≈1.00,P0.05);以09CS的定值为标准检测的89SF的11个血清型抗荚膜多糖抗体的新值与其原定值比较,各血清型的误差均20%。结论实验完成了人肺炎球菌参考血清09CS中的11个血清型抗荚膜多糖抗体IgG含量的准确定值。     

MALDI-TOF MS对肺炎链球菌鉴定与分型的应用

目的 探讨MALDI-TOF MS对肺炎链球菌鉴定和质谱分型的应用价值。方法 收集2009年1月至2013年5月温州医科大学附属第二医院临床分离的112株肺炎链球菌标本,采用Optochin敏感试验和全自动细菌分析仪对收集的菌株进行鉴定验证,并用Microflex MALDI-TOF质谱仪进行分析鉴定。根据质谱图的相似性进行细菌同源聚类树分析并构建质谱分型模型,采用荚膜肿胀试验对参与分型的菌株进行血清型比较。结果 除20株不符合检测条件之外,92株临床菌株和1株标准株经质谱分析均为肺炎链球菌,选取的60株菌株以0.5的差异水平,将60株肺炎链球菌分为18个质谱型别,在这些菌株的血清分型中有19F、19A、23F、23A、3和14六个血清型别,分布于不同的MALDI-TOF MS分型中,其中19F有18株,占30%（18/60）,分布在6种不同的MALDI-TOF MS分型中,也有3型血清型较为集中地分布于相应的MALDI-TOF MS一个型别里。结论 MALDI-TOF MS能快速、准确、简便地鉴定肺炎链球菌,且能达到种的水平。对比血清型,按照0.5差异水平,建立的18个质谱分型部分的型别与血清型有一致性,但也存有差异。     

新型肺炎链球菌疫苗研究进展

肺炎链球菌(Streptococcus pneumoniae)是引发人类肺炎、中耳炎、脑膜炎的主要病原体。为了预防肺炎链球菌感染,人类已研制出了多种相关的疫苗。目前,常用23价肺炎球菌多糖疫苗(23-valent pneumococcal polysaccharide vaccine,PPV23)预防成人和2岁以上儿童肺炎链球菌的感染,用7价、10价、13价肺炎球菌结合疫苗来预防婴幼儿肺炎链球菌的感染。虽然荚膜多糖类疫苗能预防其所包含的肺炎链球菌血清型的感染,但非疫苗血清型的菌株还是可以在鼻咽部定植,并引起感染,而且结合疫苗研制工艺复杂、价格昂贵,在发展中国家使用还不普及。鉴于上述原因许多疫苗公司开始研发具有保护力强、价格低廉的新型疫苗。近年来,肺炎链球菌全细胞疫苗和肺炎链球菌纯化蛋白疫苗由于其血清非依赖性和较低的生产成本已成为人们关注的焦点。现对肺炎链球菌新型疫苗的研制及临床应用作一概述。     

NIH小鼠模型在评价13价肺炎球菌结合疫苗免疫原性中的作用

目的 研究小鼠模型在评价13价肺炎球菌结合疫苗(pneumococcal conjugate vaccine, PCV)免疫原性中的作用。方法 15批13价PCV免疫NIH小鼠,免疫3针后采血,检测血清抗不同血清型荚膜多糖IgG抗体的含量和调理吞噬杀菌抗体的水平。检测方法采用WHO推荐的ELISA和OPA。系统性比较IgG抗体含量和调理吞噬杀菌滴度之间的相关关系,包括组内相关关系和均值相关关系以及两者之间在统计学上的差异。结果 (1)各型的组内相关性在每个组之间有所不同23F型OPA滴度与IgG抗体水平之间相关系数比较低,而且各组之间的差异均有统计学意义(P均<0.05);3型的相关系数比较高,而且与大部分血清型(如4、6A、6B、9V、14、18C、19F和23F型)之间的差异均有统计学意义(P均<0.05);而7F型和几乎所有的型(23F型除外)之间差异均无统计学意义(P均>0.05)。另外,方差分析结果表明,各型之间总体的差异有统计学意义(P<0.05)。(2)每组有13个血清型,其在各组之间的差异除了G7组与其他12组的差异有统计学意义之外,其他各组之间...     

19群肺炎链球菌国家标准菌株分子特征分析及在菌株质量控制中的应用

目的明确19群肺炎链球菌(Streptococcus pneumoniae)国家标准菌株的分子特征,为完善中国肺炎链球菌国家标准菌株的质量标准提供依据。方法在传统肺炎链球菌菌种检定方法的基础上,应用16S rRNA基因分析、聚合酶链式反应(polymerase chain reaction,PCR)血清分型、多位点序列分型(multilocus sequence type,MLST)和脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分型等多种分子生物学质控方法,对来源于中国医学细菌保藏管理中心的20株19群肺炎链球菌国家标准菌株进行分析。结果 20株19群肺炎链球菌的16S rRNA基因序列与肺炎链球菌模式株NCTC 7465的16S rRNA基因序列的相似性在99.64%～100%之间,碱基差异0～5 bp。PCR血清分型结果表明:11株19F型菌株均检测到大小为304 bp的19F型特异性扩增条带,6株19A型菌株均检测到大小为478 bp的19A型特异性扩增条带,PCR血清分型结果与血清凝集试验结果一致。MLST分型结果表明,相同血清型的菌株可以具有不同的序列型(sequence type,ST)。共获得12个ST型,其中6个ST型(10496、10497、10501、10502、10503和10509)为首次报道。PFGE分型结果显示:各型别菌株各具有其特征性PFGE带型(9～17条带),相同血清型或ST型菌株可能具有不同的PFGE带型。共存在18种不同的PFGE带型,其中19F型和19A型菌株中各有一对菌株的ST型和PFGE图谱完全一致。菌株的传代稳定性考察结果显示:在25代以内31708菌株的PFGE带型未发生变化,遗传特征是稳定的。结论 16S rRNA基因分析、PCR血清分型、MLST分型和PFGE分型等分子生物学方法应用于中国肺炎链球菌国家标准菌株的质量控制,可获得更加全面的肺炎链球菌标准菌株身份信息数据(包括序列、PCR扩增片段、序列型、图谱等),为进一步完善中国肺炎链球菌国家标准菌株的质量标准提供依据和数据支撑。     

L-Rhamnose Is Often an Important Part of Immunodominant Epitope for Pneumococcal Serotype 23F Polysaccharide Antibodies in Human Sera Immunized with PPV23

Streptococcus pneumoniae is a major human pathogen which expresses more than 90 serologically distinct capsular polysaccharides (PS) on the surface. Since pneumococcal PSs elicit protective antibodies against pneumococcal diseases, it is important to identify the immunological epitope eliciting anti-pneumococcal PS antibodies. L-rhamnose is a part of the 23F PS repeating unit and is known to be a critical part of immunodominant epitope which elicits antibodies against pneumococcal serotype 23F PS. In order to determine if L-rhamnose is a part of epitope recognized by functional antibodies specific for serotype 23F PS in human serum samples, we evaluated the opsonophagocytic killing of serotype 23F pneumococci by serum antibodies specific for L-rhamnose. Using 10 mM L-rhamnose, opsonic capacities (opsonic indices) of serum antibodies were inhibited by 60% in 19 sera (36%) and 30–60% in 16 sera (30%) out of 53 sera from young and old adults immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23). Interestingly, when IgM antibodies were depleted from immune sera in order to preferentially study IgG antibodies, the proportion of young adult sera showing more than 60% inhibition in opsonic capacity by 10 mM of L-rhamnose increased from 33% (11/31) to 68% (21/31). On the other hand, IgM depletion did not alter the proportion for old adult sera. Therefore, young and old adults may produce different antigen binding profiles of IgG antibodies against serotype 23F PS.     

PMS‐1077, a PAF antagonist,induced differentiation of HL‐60 cells with its novel activity

The cell differentiation‐inducing effect of 2‐N,N‐diethylaminocarbonyloxymethyl‐1 ‐diphenylmethyl‐4‐(3,4,5‐trimethoxybenzoyl) piperazine, hydrochloride (PMS‐1077) was determined in human leukaemic HL‐60 cells with profiling of cell proliferation, analysis of cell cycling, characterization of expression of various CD molecules and determination of phagocytotic activity of differentiated HL‐60 cells. After treatment with PMS‐1077, HL‐60 cells exhibited a decreased cell viability during which cell cycle was arrested in G₀‐/G₁‐phase. Flow cytometric analysis showed CD11b and CD14 were up‐regulated, whereas CD15 was unaffected. Together with the finding that PMS‐1077‐treated HL‐60 cells exhibited activities of differentiation by examining their ability of phagocytosing latex beads, an antiproliferative effect and a differentiation‐inducing role were determined for PMS‐1077 in HL‐60 cells.     

Opsonic and Protective Properties of Antibodies Raised to Conjugate Vaccines Targeting Six Staphylococcus aureus Antigens

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections for which a vaccine is greatly desired. Antigens found on the S. aureus outer surface include the capsular polysaccharides (CP) of serotype 5 (CP5) or 8 (CP8) and/or a second antigen, a β-(1→6)-polymer of N-acetyl-D-glucosamine (PNAG). Antibodies specific for either CP or PNAG antigens have excellent in vitro opsonic killing activity (OPKA), but when mixed together have potent interference in OPKA and murine protection. To ascertain if this interference could be abrogated by using a synthetic non-acetylated oligosaccharide fragment of PNAG, 9GlcNH₂, in place of chemically partially deacetylated PNAG, three conjugate vaccines consisting of 9GlcNH₂ conjugated to a non-toxic mutant of alpha-hemolysin (Hla H35L), CP5 conjugated to clumping factor B (ClfB), or CP8 conjugated to iron-surface determinant B (IsdB) were used separately to immunize rabbits. Opsonic antibodies mediating killing of multiple S. aureus strains were elicited for all three vaccines and showed carbohydrate antigen-specific reductions in the tissue bacterial burdens in animal models of S. aureus skin abscesses, pneumonia, and nasal colonization. Carrier-protein specific immunity was also shown to be effective in reducing bacterial levels in infected lungs and in nasal colonization. However, use of synthetic 9GlcNH₂ to induce antibody to PNAG did not overcome the interference in OPKA engendered when these were combined with antibody to either CP5 or CP8. Whereas each individual vaccine showed efficacy, combining antisera to CP antigens and PNAG still abrogated individual OPKA activities, indicating difficulty in achieving a multi-valent vaccine targeting both the CP and PNAG antigens.     

Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping

Background

Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage.

Methods

A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates.

Results

Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently identified serotypes were 11A, 13, 15B/15C, 16F and 10A.

Conclusion

The rmPCR assay performed well for the 21 serotypes/-groups included in the assay. However, in our study setting, a large proportion of serotypes were not detected by rmPCR. The sequetyping assay performed well, but did misassign specific serotypes. It may be useful for regions where vaccine serotypes are less common, however confirmatory testing is advisable.     

肺炎链球菌溶血素黏膜免疫抗定植保护作用的研究

目的原核表达△A146Ply蛋白,评价△A146Ply黏膜免疫对肺炎链球菌（Streptococcuspneumon—ioe,5．Pn）在宿主鼻咽部定植的保护作用。方法IPTG诱导、Ni—NTA树脂纯化获得纯化的△A146Ply蛋白,经黏膜免疫BALB／C小鼠,制备其特异性抗血清;进行体内抗定植实验,观察小鼠鼻咽部灌洗液和肺部残存的细菌数量,检测△A146Ply黏膜免疫对19F型肺炎链球菌在鼻咽部定植的保护作用。为验证该保护作用是否具有广谱性,培养血清型14型、3型、6型和2型S．pn,经鼻腔感染免疫后小鼠,评价△A146Ply蛋白黏膜免疫对多株肺炎链球菌定植的保护作用。进行体外抗黏附实验,检测△A146Ply蛋白及其抗血清是否对无荚膜的肺炎链球菌R6黏附A549细胞具有抑制作用。结果获得了纯度〉90％的目的蛋白;体内实验结果显示,△A146Ply黏膜免疫可以显著降低肺炎链球菌19F在宿主鼻咽部和肺部残存的细菌数量（P〈0．01）;14型和3型肺炎链球菌在免疫后小鼠鼻咽部及肺部定植的数量均显著下降（P〈0．05）,2型肺炎链球菌在免疫后小鼠肺部定植的数量显著下降（P〈0．05）,6B型肺炎链球菌在免疫组与对照组小鼠鼻咽部及肺部均无显著差异（P〉0．05）;△A146Ply特异性抗血清△A146Ply蛋白对R6黏附A549细胞的抑制效应呈剂量依赖性。结论△A146Ply蛋白经黏膜免疫BALB／C小鼠可以对多种血清型的肺炎链球菌在宿主鼻咽部及肺部的定植提供显著保护作用,为Ply作为肺炎链球菌疫苗候选蛋白的应用提供了实验依据。     

Regulation of aged humoral immune defense against pneumococcal bacteria by IgM memory B cell

Elderly persons have a high incidence of lethal infections by encapsulated bacteria. However, mechanisms involved in their poor defense and maintenance of immunological memory have been poorly understood. The present study characterized the population of B cells known as IgM memory B cell compartment and their response by pneumococcal vaccine in elderly people. CD27+ memory B cells, particularly IgD+IgM+CD27+ IgM memory B cells, had dramatically declined in the aged. Their Ig syntheses by B cells and the differentiation into plasma cells were diminished in vitro compared with those in adults. A rise of anti-pneumococcal IgM in sera of elderly persons was found with lower levels compared with those in adults after pneumococcal vaccination. Although diminished function itself of aged B cells surely exist, decline of the IgM memory B cell pool is expected to result in a poor humoral immunity against pneumococcal infection in elderly people.     

Tamarin model of pneumococcal bacteremia

Tamarins (Saguinus labiatus) were utilized to study host defenses against pneumococcal bacteremia. Tamarins had a poor antibody response to immunization with varying doses of pneumococcal capsular polysaccharide (PCP) vaccine (2 of 15 positive) or to infection with serotype 7F Streptococcus pneumoniae (2 of 14 positive). Tamarins were protected against challenge with a lethal dose of serotype 7F S. pneumoniae if the bacteria were preopsonized with human immune globulin intravenous or if the tamarins were injected with the immune globulin 30 min before challenge. There was minimal protection utilizing a mouse monoclonal anti-type 7F PCP antibody.     

An in vitro model to assess pneumococcal adherence to nasopharyngeal cells under competition conditions

Pneumococcal conjugate vaccine (PCV7) reduces invasive disease and carriage caused by vaccine serotypes (VS). An increase in carriage and disease with non-vaccine serotypes (NVS) has been observed. We have developed an in vitro model with human nasopharyngeal (NP) epithelial cells (Detroit 562) to assess the adherence capacity of Streptococcus pneumoniae to NP cells in the presence or absence of a competing Pnc strain. Two hundred and fifty pneumococcal (Pnc) strains (10 strains per serotype for 7 VS and 18 NVS) were tested for their opacity phenotype. Strains exhibiting (> or =50%) the transparent phenotype (n=72) were evaluated for their adherence capacity to Detroit 562 cells. Mean adherence capacity (> or =129 CFU/well) to NP cells was high for VS 18C, 4, and 9V and for NVS 16F, 10A, and 6A. In the in vitro competition experiments, VS strains out-competed (42/108) or co-existed (43/108) with NVS strains for adherence to NP cells in most co-inoculations. By contrast, NVS (15C, 16F, 31, and 35B) out-competed with VS in only 9 of 108 co-inoculations. Serotype 16F out-competed or co-existed with some VS and NVS strains. This model may be used to identify Pnc strains of a given serotype with competitive potentials for replacement of VS in the nasopharynx and to screen Pnc strains for animal colonization models.