【作者】 王林会；

【导师】 徐永平； Jean C.Lee；

【作者基本信息】 大连理工大学 ， 生物化工， 2013， 博士

【摘要】 金黄色葡萄球菌(简称金葡菌)是一种重要的人畜共患病病原菌,荚膜和外毒素是其重要的致病因子,本研究以这两类抗原为靶点寻找有效的免疫学手段以替代抗生素防治感染性疾病。针对奶牛乳房炎,本课题组前期研究发现,特异性卵黄抗体(IgY)具有显著的治疗作用,但是针对荚膜及外毒素的IgY尚未研究。针对人类金黄色葡萄球菌感染,目前尚无有效的疫苗。而糖工程是生产多糖-蛋白生物结合疫苗的全新技术,通过这一手段可将金黄色葡萄球菌荚膜多糖和外毒素交联制成多价疫苗,并最大程度保护其抗原决定簇。本研究考察了荚膜-毒素生物结合疫苗在人类重要感染疾病模型中的预防作用。(1)针对奶牛乳房炎,本研究采用5型(CP5)、8型(CP8)和336型(无荚膜)金黄色葡萄球菌全菌疫苗免疫蛋鸡后分离纯化获得IgY (IgY-T5, IgY-T8, IgY-T336),纯度达到80%。ELISA检测结果表明,IgY-T5、 IgY-T8及IgY-T336特异性地结合相应血清型的抗原,三者间无交叉反应,而且都不与酸奶发酵所用保加利亚杆菌结合,故对酸奶生产无不良影响。体外抑菌实验表明,在液体培养基中生长8h期间,15mg/ml的IgY-T5和IgY-T8不能抑制T5和T8荚膜金黄色葡萄球菌的生长,而15mg/ml的IgY-T336可显著抑制(4～8h)T5、T8和T336菌的生长,抑制强度约为2个Log单位,提示抑菌作用主要依赖于IgY与表面蛋白而非荚膜的结合。细菌侵袭实验表明,5mg/ml的IgY-T5, IgY-T8, IgY-T336能够分别抑制5,8和336型金黄色葡萄球菌对奶牛乳腺上皮细胞(MAC-T)的侵袭。以上结果表明,针对荚膜的特异性卵黄抗体在奶牛乳房炎的防治中具有潜在应用价值。(2)金黄色葡萄球菌外毒素也是奶牛乳房炎的一个主要致病因子,本研究分离金黄色葡萄球菌V8及RN6390的总外毒素,通过免疫蛋鸡获得特异性抗体(IgY-toxins)乳酸脱氢酶(LDH)释放实验表明,IgY-toxins可有效阻断金黄色葡萄球菌外毒素对MAC-T细胞及嗜中性粒细胞的裂解,此保护作用呈现浓度依赖性。本实验中用表达GFP的336型金黄色葡萄球菌菌株RN6390来评价IgY对金黄色葡萄球菌入侵MAC-T细胞的阻断作用。通过荧光显微镜观察,5mg/ml的IgY-T336可明显阻断RN6390对MAC-T细胞的侵袭,5mg/ml的IgY-T336和0.5mg/ml的IgY-toxins联合使用时能够更好地阻断金黄色葡萄球菌侵袭MAC-T细胞,而且细胞生长形态大大改善。临床型乳房炎实验表明,三种荚膜特异性IgY联合治疗(IgY-T5,IgY-T8及IgY-T336各20mg)5天后,患病奶牛乳液中体细胞计数(SCC)明显降低,使用20mg的IgY-toxins可达到同等效果。如果将三种荚膜特异性IgY(每种20mg)和IgY-toxins (20mg)联合使用两天后,体细胞数就得到显著降低,SCC数降至1～2×104cells/ml,并一直保持到实验的第6天,且最终SCC水平低于抗荚膜或者毒素IgY单独治疗组,疗效与青霉素(100mg)治疗组相当。通过感观评价治疗后第6天奶样发现,所有4个治疗组奶样凝块水平都明显低于对照组,组间无差异。3个IgY治疗组不能显著降低奶样中金黄色葡萄球菌数。结合(1),以上结果提示抗荚膜和毒素特异性IgY联合使用比单独使用更有效,IgY在乳房炎的治疗中应该着眼于中和毒素和抵御金黄色葡萄球菌入侵上皮细胞,而非抑菌作用。(3)针对人类的感染疾病,体外功能性实验表明,荚膜多糖-毒素生物结合疫苗免疫所得抗体αCP5-Epa、 αCP5-Hla、αCP8-Epa能特异性地调理5型和8型金黄色葡萄球菌被嗜中性粒细胞杀伤, αCP5-Hla还可有效中和α-毒素的细胞毒作用。提示,糖工程技术可在大肠杆菌中成功表达具有免疫原性和生物学功能的金黄色葡萄球菌结合疫苗。(4)金黄色葡萄球菌菌血症是临床上很常见的感染,本研究通过小鼠菌血症模型研究发现,主动免疫0.2μg CP5-2μg Epa可有效预防小鼠菌血症及体重下降、肾脏脓肿。被动免疫1mg的α CP5-Epa可有效预防Reynolds、 USA100及USA200所致的小鼠菌血症。主动免疫2(μg CP5-20μg Hla或被动免疫1mg的α CP5-Hla有效预防小鼠菌血症。对于CP8菌株,主动免疫0.2μg CP8-2μg Epa可预防Reynolds (CP8+)所致菌血症,但是对体重下降、肾脏脓肿没有显著的保护作用。被动免疫1mg的α CP8-Epa可显著预防Reynolds (CP8+)引起的小鼠菌血症。提示CP5和CP8疫苗对系统性感染-菌血症的预防切实有效。(5)金黄色葡萄球菌肺炎也是临床上非常严重的感染,致死率很高。本研究通过小鼠模型研究发现,主动免疫2μg CP5-20μg Hla可有效预防由Newman (CP5+)以及两株MRSA菌株LAC(CP-)和ST80(CP8+)所引起的致死性肺炎,同时可有效预防Reynolds(CP5+)所致小鼠菌血症。小鼠免疫CP5-Hla后,利用单克隆抗体将CD4+T细胞和CD8+T细胞剔除,其存活率与不剔除时无显著差异。提示,T细胞在CP5-Hla主动免疫保护过程中不起作用。被动免疫1mg α CP5-Hla可有效预防小鼠的菌血症,而且能够显著延长小鼠在致死性肺炎模型中的存活时间,但是不能提高存活率。分别在攻毒前24h和2h被动免疫1mg的α CP5-Hla可显著预防小鼠死于致死性肺炎。(6)金黄色葡萄球菌也能在医疗器械,如导管上定殖,甚至形成生物被膜,从而导致感染。在导管介导的大鼠心内膜炎模型中,α CP5-Hla不能起到稳定的保护作用,对模型相关的心脏瓣膜赘生物的形成、菌血症水平、体重指数、肾脏细菌水平及存活率只是呈现一定的保护趋势。抗体剂量需要进一步摸索。以上结果表明,金黄色葡萄球菌荚膜多糖和外毒素是免疫防治策略很好的靶点。因此,针对这两个靶点的IgY可有效治疗临床型金黄色葡萄球菌奶牛乳房炎。利用糖工程方法生产的荚膜-毒素生物结合疫苗可有效预防小鼠菌血症(系统性感染)、肺炎(局部性感染),而且对心内膜炎(外科介入相关感染)有一定的保护趋势。针对金黄色葡萄球菌荚膜及外毒素的免疫策略在人畜共患病的防治中具有很好的应用潜力。更多还原

【Abstract】 Staphylococcus aureus(S. aureus) is one of the most common pathogens that are responsible for the infections in both human and livestocks. And, the capsule and exotoxins of S. aureus are two major virulent factors in the pathogenisis of these infections. To develop an alternative to antibiotic therapy, this study was conducted to evaluate antibody-based immunotherapies which targeting capsule and exotoxins in bovine mastitis and human infections models, including bacteremia, pneumonia and endocarditis. Our preliminary research indicated that egg yolk antibody (IgY) has great potential to treat bovine mastitis caused by S. aureus. However, IgY targeting capsule and exotoxins were not investigated up to now. And, glycoengineering is a novel technology through which S. aureus capsular polysacharrides (CP) could be conjugated to exotoxin to creat a multivalent vaccine. In this study, the bioconjugate vaccines were evaluated in three infection models induced by multiple strains of S. aureus.(1) Capsule-specific IgY (IgY-T5, IgY-T8and IgY-T336) was obtained by immunizing laying hens with vaccines containing inactivated whole cells of serotypes5,8and336S. aueus and partially purified to80%purity. ELISA indicated that IgY binded the S. aureus cell wall specifically and showed no binding to the cell wall of Lactobacillus bulgarius, a commercially used probiotic for yorgert production. In a growth inhibition assay conducted in Columbia broth,15mg/ml of IgY-T5or IgY-T8were not able to inhibit the growth of Lowenstein or Wright strain during an8h of incubation. However, IgY-T336inhibated the growth of types5,8and336strains by about2log units in CFU. In an internalization assay,5mg/ml of specific IgYs were able to block the invasion of their homogolous strains to MAC-T cells. These results suggest that capsule-specific IgY has great potential in the treatment of bovine mastitis.(2) Exotoxins-specific IgY (IgY-toxins) was obtained by immunizing laying hens with the total toxins isolated from culture supernatant of S. aueus V8and RN6390strains. IgY-toxins was shown to protect MAC-T cells and bovine pneutrophils from lysis induced by S. aureus exotoxins, with a dose-dependent mode. And,5mg/ml of IgY-T336markly blocked the invasion of GFP-expressing RN6390to MAC-T cells. Furthermore, IgY-T336provided greater protection when combined with0.5mg/ml of IgY-toxins, at same time, improved the cell growth morphology. Treatment with20mg of IgY-T5, IgY-T8, IgY-T336each or20mg of IgY-toxins for6days significantly reduced the somatic cell counting (SCC) of milk sampled from the cow with clinical mastitis caused by S. aureus. Intramammary infusion with a combination of capsule and exotoxin specific IgYs showed an even lower level of SCC (1～2×104cells/ml), with no difference with the positive control (100mg of penicillin treatment). And, milk samples from the all treatment groups contained significantly lower level of clots compared to the PBS control. However, no difference was observed between all four treatment groups. None of the treatments, with exception of penicillin, significantly reduced the bacterial level in the milk samples. Together, these results suggest that application of IgY in the treatment of mastitis should focus on the toxin neutralizing and cell-invasion blocking activities, but not bacterial growth inhibition activity of IgY.(3) Through glycoengineering, CPs were successfully conjugated to Pseudomonas aeruginosa exotoxin A (Epa) or S. aureus a-toxin (Hla). In vitro function studies showed that a CP5-Epa, a CP5-Hla and a CP8-Epa specifically opsonized the types5or8strains for killing by human neutrophils. Besides, a CP5-Hla was able to to protect red blood cells against lysis induced by native α-toxin. These results indicated that glycoengineering effectively produced antigenic capsular polysacharrides and toxin with right epitops of S. aureus.(4) S. aureus is one of the most common pathogens responsible for bacterimia in human clinical practice. Mouse model study demonstrated that active immunization with0.2μg CP5-2μg Epa protected mice against bacteremia, body weight loss and renal abscess formation. Passive transfer with1mg of a CP5-Epa significantly reduced the bactereia level induced by S. aureus Reynolds, USA100or USA200. Active immunization with2μg CP5-20μg Hla or passive immunization with1mg of a CP5-Hla protected mice against bacteremia induced by S. aureus Reynolds (CP5+). Active immunization with0.2μg CP8-2μg Epa or passive transfer with1mg of a CP8-Epa protected mice against bacteremia induced by Reynolds (CP8+). These results indicated bioconjugate vaccine targeting CP5and CP8effectively fended off the systemic infection-bacteremia caused by S. aureus.(5) Cases of pneumonia caused by S. aureus is reported frequently and characterized with high lethallty rate. Mice immunized with2μg CP5-20μg Hla were protected against lethal pneumonia induced by Newman and MRSA strains LAC (CP-) and ST80(CP8+) and bacteremia induced by Reynolds. Depletion of CD4+or CD8+T cells in mice immunized with2μg CP5-20μg Hla did not affect the survival rate of mice intranally challenged with Newman. Passive transfer of1mg of a CP5-Hla, but not a CP5-Epa, significantly extended the survival time of mice, however, did not improve the survival rate in lethal pneumonia model. Passive immunization with two doses of1mg of a CP5-Hla at24h and2h before challenge significantly protected mice against lethal pneumonia. This phenomenon indicated that antibody, but not T cell, plays an important role in the protection against lethal pneumonia. And, Hla, but not CP, is the major virulent factor in S. aureus pneumonia model.(6) S. aureus could colonize on some medical implants, catheter for example, and form biofilm. In catheter-associated endocarditis model, passive transfer of1or15mg of a CP5-Hla showed a trend of, but not significant reduction in bactereial level in aortic valve vegetation, bacteremia, body weight loss, renal bactereial burden and survival rate.In conclusion, a combination of IgY targeting capsule and exotins of S. aureus showed great efficacy in the treatment of bovine mastitis. And, CP-toxin bioconjugate vaccines produced by glycoengineering block these two virulent factors and demonstrated protection in bacteremia (systemic infection), pneumonia (local infection) and showed a trend of protection in catheter-associated endocarditis (catheter associated infection). Together, immunotherapies targeting capsules and exotoxins of S. aureus have great potential in the treatment and prevetion of the infectious diseases in human and livestocks.更多还原