鼠伤寒杆菌外膜蛋白作为保护性疫苗的初步实验研究

本文采用超声波破碎和Triton X-100处理、超速离心的方法,提取了鼠伤寒杆菌的外膜蛋白(OMP)。用提取的OMP免疫家兔和小鼠,经用ELISA方法测定小鼠、家兔及鼠伤寒意染患者血清中抗-OMP抗体含量,并用小鼠作足垫肿胀实验及主动和血清被动保护试验。结果免疫动物及患者血清中都含有较高滴度抗体,免疫小鼠足垫出现明显地DTH反应。50ug OMP免疫小鼠可保护500 LD50毒株的攻击,0.2ml免疫血清亦能够被动保护以上同样的毒株攻击。这些结果表明,提取的OMP有较强的免疫原性和明显的免疫保护作用,应用Western blot分析免疫血清均能识别36KD蛋白带,36KD蛋白带可能是鼠伤寒杆菌的主要免疫原。     

鼠伤寒杆菌主要外膜蛋白作为保护性抗原的研究

采用超声破碎,ＴｒｉｔｏｎＸ─１００处理和Ｓｅｐｈａｃｒａｌ超细Ｓ─３００凝胶过滤技术提取了鼠伤寒杆菌的主要外膜蛋白（ＭＯＭＰｓ）。ＭＯＭＰｓ的脂多糖（ＬＰＳ）含量约为０．２％。经ＳＤＳ─ＰＡＧＥ图谱显示蛋白在３６─４１ＫＤ之间。ＭＯＭＰｓ能使小鼠产生典型的足垫肿胀（ＤＴＨ）及高水平的ＩＬ─２;可保护５００ＬＤ５０鼠伤寒杆菌及伤寒杆菌的攻击,其免疫保护率分别为９０％和３３．３％,用５０ｕｇＭＯＭＰｓ免疫的小鼠的Ｔ淋巴细胞经尾静脉注射给非免疫小鼠,可使后者得到被动免疫保护,其保护率为４２．９％。基于上述实验结果,本文认为鼠伤寒杆菌的ＭＯＭＰｓ是一良好的保护性抗原。     

伤寒杆菌SOD抗体对小鼠腹腔吞噬细胞杀灭伤寒杆菌的影响

为了制备伤寒要直菌ＳＯＤ抗体,研究该抗体对小鼠腹腔吞噬细胞杀灭伤寒杆菌的影响,探讨ＳＯＤ与伤寒杆菌抗吞噬作用的关系。采用纯化的伤寒杆菌（Ｓｔｗｌ）Ｆｅ－ＳＯＤ免疫农兔制备兔抗Ｆｅ－ＳＯＤ抗体。利用ＥＡＬＩＳＡ法检测抗体的效价。免疫电泳、双向琼脂扩散试验和抗体对酶活性抑制试验分析抗体的特性。用不同浓度的抗体预先处理伤寒杆菌,然后进行小鼠腹腔吞噬细胞的吞噬杀菌试验。试验结果抗体的ＥＬＩＳ攻疚价为１：１     

IL-12增强流感血凝素DNA疫苗在小鼠中抗流感作用

流感病毒的表面抗原血凝素 (hemagglutinin ,HA)能作为DNA疫苗抗流感病毒攻击 ,在小鼠模型中检测白介素 12 (interleukin 12 ,IL 12 )能否作为HADNA疫苗佐剂增强小鼠抗流感病毒攻击。将IL 12和HA共同免疫小鼠 ,免疫 2次 ,间隔 3周 ,加强免疫后用致死量流感病毒攻击。共同免疫IL 12和HADNA与单独免疫HA相比 ,无论初免还是加强免疫后血清中抗HA的IgG抗体显著提高 ,小鼠体重丢失 (一种临床症状 )明显减少且提高了小鼠的存活率。这些结果表明了IL 12能作为一种佐剂提高流感DNA疫苗的免疫效价。     

表达乙型脑炎病毒抗原的非复制型重组痘苗病毒能在小鼠中诱发免疫保护

使用2×107PFU乙脑病毒野毒株GSS抗原preM-E-NS1-NS2a的非复制型重组痘苗病毒NTV-JEV免疫3周龄BALB/c小鼠,小鼠体内乙脑病毒特异性抗体滴度1∶71,IgG2a/IgG1为1.5,中和抗体滴度为1∶8,免疫组小鼠产生特异性补体介导的细胞杀伤作用。以10 LD50乙脑P3株病毒对小鼠进行了脑内攻击,NTV-JEV免疫组小鼠存活率为100%,与乙脑减毒活疫苗SA14-14-2免疫组存活率相同。100 LD50P3株病毒攻击后NTV-JEV免疫组存活率28.6%,与SA14-14-2组免疫效果无明显差别。存活小鼠体内乙脑病毒特异性抗体升高为1∶179,IgG2a/IgG1比值为1.9,中和抗体大于1∶16。以30 LD50GSS株病毒进行攻击后,NTV-JEV免疫组和SA14-14-2组保护率均为8.3%。上述结果表明,NTV-JEV与乙脑减毒活疫苗SA14-14-2株具有相近的保护力,NTV-JEV不仅可以保护小鼠抵抗同株病毒GSS的攻击,而且可以抵抗异株病毒P3的攻击。     

鼠伤寒沙门氏菌外膜蛋白诱发BALB／C小鼠免疫保护的研究

本文采用超声破碎ＴｒｉｔｏｎＸ—１００和超速离心技术提取了鼠伤寒杆菌（Ｓａｌｍｏｎｅｌｌａｔｙｐｈｉｍｕｒｉｕｍ,ＳＴＭ）的外膜蛋白（Ｏｕｔｅｒｍｅｍｂｒａｎｅｐｒｏｔｅｉｎｓ,ＯＭＰｓ）,并发现ＯＭＰｓ中脂多糖ＬＰＳ的含量约为５％,ＯＭＰｓ经ＳＤＳ—ＰＡＧＥ显示１０余条蛋白带。对ＯＭＰｓ诱发ＢＡＬＢ／Ｃ小鼠产生典型迟发型变态反应ＤＴＨ和ＩＬ—２的水平进行了检测。经腹腔免疫的小鼠用５００ＬＤ５０鼠伤寒杆菌（５０１１５）攻击,１００％可得到保护;用５００ＬＤ５０伤寒杆菌（Ｅ６８６）攻击,３３．３％可得到交叉保护。免疫ＢＡＬＢ／Ｃ小鼠的Ｔ淋巴细胞,经尾静脉注射给非免疫小鼠,可使后者得到８５．７％的被动免疫保护,上述结果说明ＯＭＰｓ能诱发ＢＡＬＢ／Ｃ小鼠细胞免疫和保护性免疫,并提示成为分子疫苗的可能性。     

季节性流感裂解疫苗在小鼠中针对同型与异型流感病毒的免疫保护效力

为了研究季节性流感裂解疫苗在小鼠中针对甲型流感病毒同型同株、同型异株、异型异株攻击的免疫保护效力及其与诱发的血凝抑制(HI)抗体滴度的关系,本研究使用我国2008~2009年度季节性流感裂解疫苗中不同剂量的甲1型流感病毒H1N1(疫苗株病毒A/Brisbane/59/2007(H1N1)-like)和甲3型流感病毒的H3N2(疫苗株病毒A/Brisbane/10/2007(H3N2)-like)疫苗组分免疫BALB/c小鼠,首先确定了能在小鼠中诱发血HI抗体滴度达到40的疫苗免疫剂量;然后以此剂量免疫小鼠,分别使用同型同株流感病毒(鼠肺适应株A/Brisbane/59/2007(H1N1)-like virus(MA))(简称A1)和同型异株流感病毒(鼠肺适应株A/Purto Rico/8/34(H1N1))(简称PR8)攻击H1N1疫苗免疫小鼠,使用异型异株流感病毒A1攻击H3N2疫苗免疫小鼠,通过体重变化和存活率情况,探讨季节性流感疫苗在小鼠中针对甲型流感病毒同型同株、同型异株、异型异株攻击的保护效力。结果显示,季节性流感裂解疫苗H1N1和H3N2组分按照HA不同剂量0.15μg、0.5μg、1.5μg、5μg和15μg免疫小鼠后,所诱发的HI抗体滴度随免疫剂量的增加而增强,1.5μgHA即可以诱发免疫小鼠HI抗体滴度达到40;以此剂量免疫小鼠,分别使用3LD50、10LD50、30LD50、100LD50、300LD50、1 000LD50和3 000LD50的同型同株流感病毒A1进行攻击,1.5μgH1N1疫苗可以100%保护小鼠抵御高至1000LD50同型同株流感病毒A1的攻击,15μg甚至可以100%保护3 000LD50同型同株流感病毒A1的攻击,但是这两个剂量免疫的小鼠在低至3LD50同型异株流感病毒PR8的攻击后都全部死亡;使用可以诱发HI抗体滴度达到140的15μg H3N2疫苗免疫小鼠,在低至3LD50异型异株流感病毒A1的攻击后亦全部死亡。以上结果表明,季节性流感疫苗可使小鼠HI抗体滴度达到40的疫苗免疫剂量为1.5μg,该免疫剂量可以有效保护小鼠抵御同型同株流感病毒的攻击,但是难以保护小鼠抵御同型异株与异型异株流感病毒的攻击,这一结果为建立以季节性流感疫苗为参考的免疫保护评价体系提供了实验依据。     

脂多糖模拟肽13L诱导对小鼠内毒素休克的保护反应

为研究LPS 2630表位模拟肽13L是否能诱导抗脂多糖（LPS）抗体的产生、对细菌攻击及内毒素休克的保护性反应,合成了模拟LPS表位的短肽13L．用合成肽13L-蓝载体（blue carrier,BC）交联物免疫Balb／C小鼠,观察小鼠体内抗LPS抗体产生的规律,并观察免疫小鼠细菌感染存活时间,用颈总动脉插管测定技术观察免疫小鼠内毒素休克的血压变化．结果显示,合成肽13L-BSA交联物能与兔抗鼠伤寒和大肠杆菌LPS抗血清及抗鼠伤寒LPS单抗结合,证明短肽13L可模拟LPS的抗原性．用13L-BC交联物免疫可诱发小鼠体内针对大肠杆菌LPS 2630和鼠伤寒沙门氏菌LPS 7261的抗体反应,该反应可被灭活大肠杆菌及两种LPS所加强,其抗体亚类主要为IgG2a,其次为IgG2b与IgM,而IgG1与IgG3含量极低．免疫13L-BC小鼠显示对细菌攻击的抵抗,与免疫BC对照小鼠比较,其存活时间分别为（12±1．3）天和（5．3±0．4）天（P〈0．01）．免疫13L-BC小鼠对静脉注射LPS诱发内毒素休克的保护作用体现在血压下降幅度明显低于对照动物,在LPS 2630攻击后1、2、3及4h时两组动物血压明显差别（P〈0．05、P〈0．01、P〈0．05及P〈0．01）,而LPS 7261攻击后1、2、3及4h时两组动物血压差别则略小于LPS 2630组（P〉0．05、P〈0．05、P〈0．05及P〈0．01）．上述结果证明,LPS表位模拟肽13L交联物免疫小鼠可产生针对细菌攻击及内毒素休克的保护性效应,提示该模拟肽作为LPS交叉保护性疫苗候选的可行性．     

C型肉毒毒素的制备及类毒素保护力初探

将C型肉毒梭菌经适宜条件的产毒培养后纯化,并进行相关鉴定。制备的C型肉毒毒素用分段脱毒法脱毒,并进行类毒素保护力的初步研究。以不同蛋白含量C型肉毒类毒素免疫小鼠后攻毒,结果显示,蛋白含量为0.625μg的类毒素免疫2针或蛋白含量为1.25μg的类毒素免疫1针均可保护50LD50的C型肉毒毒素攻击。蛋白含量为5μg的C型肉毒类毒素与福氏不完全佐剂配制的抗原免疫小鼠3次所得抗血清的保护力(Anti LD50/ml)为4.3×104。说明用该纯化工艺制备的C型肉毒类毒素具有很好的免疫原性,作为抗原成分用于C型肉毒疫苗和C型肉毒抗毒素的研究和生产具有较好的应用潜力。     

嗜水气单胞菌佐剂灭活苗免疫中华鳖抵抗嗜水气单胞菌的致死性感染

通过建立中华鳖浸泡感染模型评价中华鳖嗜水气单胞菌油乳剂灭活疫苗对中华鳖的免疫保护效力。在主动免疫保护试验中,疫苗免疫组中华鳖能产生较高的抗体水平,在使用10×LD50的嗜水气单胞菌T3株进行浸泡攻毒后,保护率为100%(10/10),生理盐水对照组中华鳖的存活率仅为30%(3/10)。在被动免疫保护试验中,疫苗腹腔免疫异育银鲫抗血清能80%(8/10)保护中华鳖抵抗10×LD50的T3株的腹腔接种的攻击,生理盐水对照组中华鳖的存活率为20%(2/10)。研究结果表明嗜水气单胞菌佐剂油乳剂灭活苗具有良好的免疫学原性,可有效预防由嗜水气单胞菌引起的中华鳖红底板和肠道败血症等疾病。     

Recall of Immunity in Mice Vaccinated with Salmonella enteritidis or Salmonella typhimurium

Mice were immunized with living Salmonella enteritidis or S. typhimurium and then were reinfected 8, 30, 60, and 150 days later with streptomycin-resistant strains of S. enteritidis, S. typhimurium, or a mixture of the two organisms. The level of resistance at the time of challenge and the rate at which resistance was recalled in late convalescence was determined by daily liver and spleen counts of the challenge population. An immediately effective specific antibacterial immunity was maintained only while the vaccinating strain could still be detected in the liver and spleen. Reinfection of vaccine-free mice with the homologous organism caused a rapid recall of antibacterial immunity (within 3 days), but the response to the heterologous organism was much slower (5 to 8 days). Simultaneous injection of both pathogens into the vaccinated mice resulted in liver and spleen growth curves which resembled those obtained when the two organisms were administered separately. The implications of these growth studies in the development of specific cellular immunity to Salmonella infections are discussed.     

Mutagenicity of oxaspiro compounds with Salmonella

The spiro attachment of an epoxide group to a tetrahydropyran ring in the trichothecene mycotoxins has prompted this study of the mutagenicity and alkylation rates of the trichothecene, anguidine, and 5 related model oxaspiro compounds. While the model compounds were weak alkylating agents of 4-(4-nitrobenzyl)pyridine as a test nucleophile, anguidine lacks such activity. Also, while mutagenicity was not established for anguidine in Salmonella TA100, 3 of the oxaspiro compounds were weakly mutagenic and 2 compounds were toxic to the bacteria. The toxicity and mutagenicity of the model compounds are more related to their polarity than to their alkylation rates.     

Egg contamination by Salmonella serovar enteritidis following vaccination with Delta-aroA Salmonella serovar typhimurium

The efficacy of an aroA Salmonella serovar typhimurium modified live vaccine to decrease internal egg contamination after oral challenge of hens with egg-contaminating Salmonella serovar enteritidis was assessed. Challenge was with a mixed phenotype of S. enteritidis that had virulence characteristics previously associated with enhanced oral invasiveness and egg contamination in chickens. Immunized birds had fewer positive ovary/oviduct pools and lower cfu g(-1) cecal contents than did non-immunized birds, but the differences were not significant. The number of positive intestinal (duodenum, jejunum, ileum) and organ (spleen, kidney, liver) pools following challenge from each treatment group were equivalent. Most importantly, immunization did not decrease egg contamination. These results suggest that the ability of modified live vaccines to reduce internal egg contamination by S. serovar enteritidis can be assessed using characterized strains for challenge.     

Invasive Illness with Salmonella Virchow Infection

Salmonella virchow infection in man is usually sporadic and is little known in Britain. We report an outbreak of 21 cases in the Manchester area. The source of infection was believed to be chickens bought at different shops. Symptoms were typhoidal or septicaemic.     

Salmonella paratyphi C: Genetic Divergence from Salmonella choleraesuis and Pathogenic Convergence with Salmonella typhi

Background

Although over 1400 Salmonella serovars cause usually self-limited gastroenteritis in humans, a few, e.g., Salmonella typhi and S. paratyphi C, cause typhoid, a potentially fatal systemic infection. It is not known whether the typhoid agents have evolved from a common ancestor (by divergent processes) or acquired similar pathogenic traits independently (by convergent processes). Comparison of different typhoid agents with non-typhoidal Salmonella lineages will provide excellent models for studies on how similar pathogens might have evolved.

Methodologies/Principal Findings

We sequenced a strain of S. paratyphi C, RKS4594, and compared it with previously sequenced Salmonella strains. RKS4594 contains a chromosome of 4,833,080 bp and a plasmid of 55,414 bp. We predicted 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) and 152 pseudogenes (149 in the chromosome and 3 in the plasmid). RKS4594 shares as many as 4346 of the 4,640 genes with a strain of S. choleraesuis, which is primarily a swine pathogen, but only 4008 genes with another human-adapted typhoid agent, S. typhi. Comparison of 3691 genes shared by all six sequenced Salmonella strains placed S. paratyphi C and S. choleraesuis together at one end, and S. typhi at the opposite end, of the phylogenetic tree, demonstrating separate ancestries of the human-adapted typhoid agents. S. paratyphi C seemed to have suffered enormous selection pressures during its adaptation to man as suggested by the differential nucleotide substitutions and different sets of pseudogenes, between S. paratyphi C and S. choleraesuis.

Conclusions

S. paratyphi C does not share a common ancestor with other human-adapted typhoid agents, supporting the convergent evolution model of the typhoid agents. S. paratyphi C has diverged from a common ancestor with S. choleraesuis by accumulating genomic novelty during adaptation to man.