C群脑膜炎球菌结合物小鼠免疫原性的研究

目的比较C群脑膜炎球菌多糖蛋白质结合物(简称结合物)的相对分子质量大小和免疫剂量对其在小鼠体内免疫原性的影响,为结合物的分子大小的质控和结合疫苗成品免疫剂量的选择提供实验依据。方法利用CDAP活化法制备C群脑膜炎球菌结合物,通过硫酸铵盐析进行纯化,然后利用Sepharose CL-4B凝胶过滤层析分析,并根据化学检测结果将结合物分为KD0-0.2(组分1)、KD0.2-0.4(组分2)、KD0.4-0.7(组分3)等3个不同相对分子质量组分,每份分别采用1.0μg、0.2μg的免疫剂量免疫小鼠,并对血清进行ELISA分析。结果采用1.0μg免疫剂量时,3种不同相对分子质量的C群脑膜炎球菌结合物均能产生较高水平的抗体,具有典型的加强效应,第2剂免疫即可产生高水平的抗体,而第2、3剂免疫后的抗体水平差异无统计学意义;采用0.2μg免疫剂量时,三种不同相对分子质量的C群脑膜炎球菌结合物只有在第3剂免疫后才能产生较高水平抗体,第1、2剂免疫后的抗体水平差异无统计学意义,组分1和组分2在第3剂免疫后产生的抗体水平优于组分3;对于相同相对分子质量结合物,用1.0μg免疫小鼠产生的抗体水平优于0.2μg免疫组,而且有显著的统计学意义。结论高剂量时,多糖相对分子质量大小对C群脑膜炎球菌结合物的免疫原性没有显著性影响;低剂量时,小相对分子质量结合物对其免疫原性有影响。同等相对分子质量时,1.0μg比0.2μg的免疫剂量可以激发更高的抗体水平。     

不同剂量肺炎链球菌荚膜多糖与蛋白结合物的免疫原性比较

用肺炎链球菌19F型、23F型的荚膜多糖(C-PS)分别和破伤风类毒素(TT)蛋白结合,制备了两个型别的多糖-蛋白结合物(19F-TT,23F-TT)。为探讨结合物的最适免疫剂量,以10μg多糖和含1μg、3μg、9μg、27μg多糖的结合物经腹腔免疫NIH小鼠,ELISA方法检测小鼠血清中多糖和含不同多糖的结合物所产生的特异性IgG抗体。结果在不同的剂量免疫小鼠后,3μg剂量组在免疫三针后可诱生高浓度的抗体,但随着免疫剂量的增加,9μg与27μg诱生的抗体浓度较3μg诱生的抗体浓度之间并无显著性差异。含多糖3μg的19F型和23F型多糖蛋白结合物剂量组可诱生高浓度的特异性IgG抗体。     

四价流感病毒裂解疫苗安全性及免疫原性研究

目的探讨四价流感病毒裂解疫苗(quadrivalent influenza vaccine,QIV)的安全性和有效性。方法用WHO当年推荐的A1型、A3型和2株B型流行性感冒病毒株制备QIV,按照企业注册标准及《中华人民共和国药典》2015版(三部)的要求全面检定后进行毒理试验和免疫原性试验。结果毒理试验结果显示,QIV在动物中未见有害作用的剂量为15μg/株;免疫原性试验结果显示,免疫效果完全能够达到或超过三价流感病毒裂解疫苗(trivalent influenza vaccine,TIV)的免疫效果,且QIV所特有的B2亚型,除最低剂量外,小鼠的血凝抑制抗体(hemagglutination inhibition,HI)滴度均超过了1∶40的阳性水平。结论本试验条件下QIV在动物中具有良好的安全性和免疫原性。     

禽波氏杆菌外膜蛋白的提取及其免疫原性的检测

为研究禽波氏杆菌OMP的免疫原性,试验采用超声波破碎、TritonX-100处理技术提取了禽波氏杆菌OMP,采用Bradford方法测定禽波氏杆菌OMP含量,进行了SDS-PAGE检测,然后制备油乳剂OMP免疫抗原,对1日龄雏鸡分别以0.3mL(OMP90μg)、0.5mL(OMP150μg)、0.8mL(OMP240μg)的剂量颈部皮下接种。结果:禽波氏杆菌OMP含量为300μg/mL;禽波氏杆菌OMP最佳免疫剂量为0.5mL/只;通过免疫抗体与攻毒保护相关性测试,抗体效价在1:28以上能抵抗致死量禽波氏杆菌的攻击。据间接ELISA法检测的抗体水平可知,抗体的持续时间足以保护雏鸡避过易感日龄,试验发现OMP具有良好的免疫原性。本试验结果将为禽波氏杆菌OMP单克隆抗体的制备、快速诊断试剂盒的研制、亚单位疫苗的开发奠定良好的基础。     

A群奈瑟氏脑膜炎球菌荚膜多糖-破伤风类毒素结合疫苗的制备及其免疫原性研究

采用溴化氰(CNBr)活化多糖,以无水己二酸二肼(ADH)作为连接剂,1乙基13(3二甲基氨基丙基)碳化二亚胺(EDAC)为偶联剂制备A群奈瑟氏脑膜炎球菌荚膜多糖(GAMP)与破伤风类毒素(TT)的结合物,经皮下免疫NIH小鼠,用ELISA检测小鼠血清中抗GAMP及抗载体蛋白的IgG抗体水平。用补体介导的体外杀菌试验检测血清中GAMP抗体的杀菌活性。结果显示,实验中制备的多糖衍生物和多糖蛋白质结合物都具有GAMP抗原特异活性。结合物免疫小鼠后可诱生比多糖单独免疫更高水平的GAMP血清IgG抗体,并能形成免疫记忆,产生再次应答。结合物免疫小鼠所诱生的血清GAMP抗体较之多糖组具有更强的体外杀菌活性。表明此方法制备的结合物可获得优于多糖的、稳定的特异免疫原性。     

甲型H1N1流感病毒裂解疫苗的免疫原性和安全性

目的评价甲型H1N1流感病毒裂解疫苗(简称甲型H1N1流感疫苗)的免疫原性和安全性。方法按照随机、双盲、安慰剂对照的原则,采用0、21天免疫程序,选择3岁及3岁以上健康者1 202人。分组为3~11岁、12~17岁、≥60岁组,按照人数基本为1∶1的比例随机分别接种7.5μg和15.0μg甲型H1N1流感疫苗;18~59岁组按照人数基本为1∶1∶1的比例随机分别接种7.5μg、15.0μg甲型H1N1流感疫苗和安慰剂对照。观察各组接种后的不良反应率以及免疫前后血凝抑制(HI)抗体阳转率、保护率、GMT水平和平均增长倍数。结果受试对象的安全性结果显示7.5μg和15.0μg组不良反应发生率分别为8.74%(48/549)和13.88%(74/533),其中Ⅱ级反应率分别为0.36%(2/549)和1.13%(6/533),未观察到Ⅲ级及以上不良反应和其他异常反应及严重不良事件。2剂接种未见不良反应叠加现象。7.5μg或15.0μg试验疫苗首剂免疫后,血清抗体阳性率分别为85.13%(395/464)和90.77%(413/455),保护率分别为85.56%(397/464)和91.43%(416/455),抗体GMT较免疫前分别增长36.1倍和52.6倍。2剂免疫后,血清抗体阳性率分别是97.84%(454/464)和99.12%(451/455),保护率分别是98.06%(455/464)和9 9.56%(453/455),抗体GMT较免疫前分别增长63.3倍和96.0倍。4个年龄组(3~11岁、12~17岁、18~59岁及≥60岁年龄组)7.5μg和15.0μg组HI抗体阳性率和保护率均大于70%,GMT较免疫前均增长2.5倍以上,结果显示7.5μg和15.0μg甲型H1N1流感疫苗接种1剂后抗体水平已达到研究方案中设定的预期标准,免疫2剂后抗体阳性率和抗体水平明显提高。结论临床试验表明甲型H1N1流感疫苗具有良好的安全性和免疫原性,且接种1剂15.0μg甲型H1N1流感疫苗,即可在3岁和3岁以上人群中产生良好的免疫效果。     

6A型肺炎球菌结合物分子大小对小鼠免疫原性的影响

目的 比较不同分子大小的6A型肺炎球菌(serotype 6A Streptococcus Pneumoniae )结合物和佐剂吸附在小鼠体内免疫原性的影响。方法 通过乙酸水解降低6A型荚膜多糖的相对分子质量制备成水解物,水解物经1-氰基-4-二甲胺基吡啶四氟硼酸盐(CDAP)活化并与破伤风类毒素己二酸酰肼衍生物TT AH 结合,制备成结合物。用Sepharose 4 Fast Flow 纯化结合物,并根据化学检测结果将结合物分为 K D 0.0~0.2、 K D 0.2~0.4、 K D 0.4~0.6等3个组分,每个组分分别以磷酸铝佐剂吸附,将吸附前后的各个组分按照每针次每只小鼠0.2 μg分别免疫小鼠,并采用ELISA检测结合物在小鼠体内的抗体水平。结果 3种不同相对分子质量吸附前后的结合物在小鼠体内均能产生较高水平的抗体,各组2、3针之间具有明显的加强效应。在吸附组和未吸附组中,3种不同分子大小的结合物在小鼠体内产生抗体水平无明显差异。各组分佐剂吸附后的结合物血清抗体滴度高于未吸附组,但这种差异无统计学意义( P > 0.05)。结论 结合物的分子大小对小鼠体内抗体水平的产生没有明显影响;磷酸铝佐剂吸附对于不同分子大小的结合物在小鼠体内的免疫原性有一定的增强效应,但这种增强效应差异无统计学意义。     

肺炎链球菌18C型糖蛋白结合物的制备及其免疫原性

制备肺炎链球菌18C型荚膜多糖－破伤风类毒素结合物（CPS－TT）,测定结合物的理化性质,抗原特异性及其在动物中的免疫原性。结果显示,结合物能与相应的多糖和破伤风抗血清形成明显的沉淀线,蛋白／多糖比率为1．86,结合物分子大小（Kd值）为0．058。注射小鼠后可诱导明显的抗体应答,而且随着注射针次的增加,抗体反应水平明显增高,显示加强效应。结果表明,制备的肺炎链球菌糖蛋白结合物抗原性良好,具有胸腺依赖性的特性,在小鼠中显示较好的免疫原性。     

福氏2a痢疾杆菌O—特异性多糖与破伤风类毒素结合疫苗的制备及其免疫学特性

通过己二酸二肼（ADH）将福氏2a痢疾杆菌脂多糖（LPS）经酸水解脱毒纯化后得到的O－SP和破伤风类毒素（TT）蛋白共价结合,制备了福氏2a痢疾杆菌结合物。以2.5μg多糖或含2.5μg多糖的结合物经皮下注射免疫NIH品系雌性小鼠,同时以25μg多糖或含25μg多糖的结合物经耳缘静脉注射家兔。用ELISA方法分别检测小鼠和家兔血清中抗脂多糖抗体水平及抗TT水平,并用豚鼠血清补体介导进行了体外杀菌实验。结果显示：用本实验方法提取的多糖,合成的多糖衍生物,多糖－蛋白结合物都具有福氏2a痢疾杆菌O－抗原特异性,其化学组成及结构特异性和国外文献报道基本一致。单独用多糖免疫小鼠和家兔未能诱导LPS抗体,而结合物免疫小鼠和家兔的血清诱导出了较高的抗LPS IgG抗体及抗TT抗体,并有较强的体外杀菌活性。产生的抗体存在着免疫记忆性,可以产生再次免疫应答加强反应。     

百日咳鲍特菌脂寡糖的制备及寡糖结合物在小鼠体内免疫原性研究

目的提取纯化百日咳鲍特菌(简称百日咳杆菌)脂寡糖(lipo-oligosaccharides,LOS),制备百日咳杆菌多糖蛋白结合物,并分析其理化性质及免疫原性。方法通过改良热酚水法提取百日咳杆菌的LOS,采用酶解法纯化LOS,通过1%冰醋酸水解获得寡糖(oligosaccharides,OS),产物经Tricine-SDS-PAGE及核磁共振氢谱(~1H NMR)鉴定。OS的羟基经1-氰基-4-二甲氨基吡啶四氟硼酸酯(1-cyano-4-dimethylamino pyridinium tetrafluoroborate,CDAP)活化后,与破伤风类毒素己二酸酰肼衍生物(TT_(AH))共价结合,制备多糖蛋白结合物,经Sephacryl S-300层析柱纯化后收集结合物原液,并对结合物的理化性质、抗原性进行分析,采用两种剂量(2.5μg/剂和5.0μg/剂)免疫BALB/c小鼠,分析其免疫原性。结果纯化后LOS纯度90%,核磁共振结果表明OS结构正确,功能基团得到保留。制备的结合物抗原性未发生改变,经3针免疫后,两种剂量下结合物均有剂次加强效应(P0.05),且两者差异无统计学意义(P0.05)。结论将百日咳杆菌OS与TT_(AH)结合是制备百日咳杆菌多糖蛋白结合疫苗的可行途径,并且结合物在小鼠体内具有良好的免疫应答。     

Evaluation of monophosphoryl lipid A (MPL) as an adjuvant. Enhancement of the serum antibody response in mice to polysaccharide-protein conjugates by concurrent injection with MPL

Concurrent injection of monophosphoryl lipid A (MPL) in saline or as an oil-in-water emulsion enhanced both the primary and secondary serum antibody responses to the capsular polysaccharide (CP) components of seven conjugates: the enhanced responses were Ag-specific. In contrast, MPL did not enhance the serum antibody response to five of the six unconjugated CP. MPL and trehalose dimycolate injected concurrently with the unconjugated Vi CP of Salmonella typhi (Vi) enhanced the serum antibody response to that Ag. MPL further enhanced the Vi antibody levels when injected with conjugates of this CP. The serum antibody responses to Pseudomonas aeruginosa exotoxin A, used as the carrier protein for the Staphylococcus aureus types 5 and 8 conjugates, were also enhanced by MPL. MPL in oil-in-water emulsion was generally more effective than when administered in saline.     

Protective efficacy and immunogenicity of Vi-porin conjugate against Salmonella typhi.

A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyl dithio)-propionate (SPDP). After preparing the Vi-porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5-fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti-Vi antibody (IgG) levels (P < 0.01) than the mice immunized with Vi alone. Anti-porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate-immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.     

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.     

Factors Affecting Formation of Incomplete Vi Antibody in Mice.

Gaines, Sidney (Walter Reed Army Institute of Research, Washington, D.C.), Julius A. Currie, and Joseph G. Tully. Factors affecting formation of incomplete Vi antibody in mice. J. Bacteriol. 90:635-642. 1965.-Single immunizing doses of purified Vi antigen elicited complete and incomplete Vi antibodies in BALB/c mice, but only incomplete antibody in Cinnamon mice. Three of six other mouse strains tested responded like BALB/c mice; the remaining three, like Cinnamon mice. Varying the quantity of antigen injected or the route of administration failed to stimulate the production of detectable complete Vi antibody in Cinnamon mice. Such antibody was evoked in these animals by multiple injections of Vi antigen or by inoculating them with Vi-containing bacilli or Vi-coated erythrocytes. The early protection afforded by serum from Vi-immunized BALB/c mice coincided with the appearance of incomplete Vi antibody, 1 day prior to the advent of complete antibody. Persistence of incomplete as well as complete antibody in the serum of immunized mice was demonstrated for at least 56 days after injection of 10 mug of Vi antigen. Incomplete Vi antibody was shown to have blocking ability, in vitro bactericidal activity, and the capability of protecting mice against intracerebral as well as intraperitoneal challenge with virulent typhoid bacilli. Production of incomplete and complete Vi antibodies was adversely affected by immunization with partially depolymerized Vi antigens.     

A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+)), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+) colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+) and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+) resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(-) infected animals. C5.507 Vi(+) infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-). The modulating effect associated with Vi was not observed in MyD88(-/-) and was reduced in TLR4(-/-) mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.     

Immunological response of three mouse strains to typhoid vaccine and Vi antigen

Vi-agglutinin, active cutaneous anaphylaxis and protective responses (ed(50)) of three mouse strains (CFW, NIH, and Balb/cAnN) to acetone-inactivated typhoid vaccine and soluble Vi antigen were compared. Seven days after immunization with either typhoid vaccine or Vi antigen the three strains of mice differed with respect to Vi-antibody titers. Significant differences were observed in the protective responses. Each mouse strain was significantly better protected by the intraperitoneal than by subcutaneous route of immunization. Active cutaneous anaphylaxis was more pronounced in showing strain differences in response to Vi antigen. The serological responses to Vi antigen of the strains of mice did not correlate with their protective response.     

伤寒Vi多糖结合疫苗免疫小鼠后的抗体类型及动态分析

伤寒Vi多糖结合疫苗和Vi多糖疫苗分别免疫小鼠,分离血清,采用间接ELISA法测定不同时点血清中特异性IgA、IgM、IgG及其亚类(IgG1、IgG2a、IgG3)的抗体滴度。结果显示,免疫一针后,Vi多糖结合疫苗组的IgG抗体GMT值明显升高,第二针有加强效应(P<0.01);所测3种IgG亚型中IgG2a抗体滴度升高明显;Vi多糖和结合疫苗免疫小鼠后,血清中IgA和IgM抗体滴度均有显著升高,但无加强应答。显示Vi多糖结合疫苗在诱导小鼠血清IgG应答方面有加强效应。     

伤寒─鼠伤寒重组株Vi4072的效力试验研究

伤寒─鼠伤寒重组株Ｖｉ４０７２所产生Ｖｉ抗原以伤寒Ｔｙ２株所产Ｖｉ抗原作对照,通过ＥＤ５０测定和小白鼠被动保护试验作了比较,结果表明Ｖｉ４０７２株Ｖｉ抗原的半数有效剂量为０．０１３６μｇ,Ｔｙ２株的半数有效剂量为０．０１８３μｇ,说明Ｖｉ４０７２─Ｖｉ对小白鼠的保护作用不低于Ｔｙ２─Ｖｉ。被动保护试验证明,在同等条件下,两种Ｖｉ抗原的免疫血清对小白鼠提供的保护作用相同。     

伤寒─鼠伤寒重组株Vi4072的效力试验研究

伤寒─鼠伤寒重组株Ｖｉ４０７２所产生Ｖｉ抗原以伤寒Ｔｙ２株所产Ｖｉ抗原作对照,通过ＥＤ５０测定和小白鼠被动保护试验作了比较,结果表明Ｖｉ４０７２株Ｖｉ抗原的半数有效剂量为０．０１３６μｇ,Ｔｙ２株的半数有效剂量为０．０１８３μｇ,说明Ｖｉ４０７２─Ｖｉ对小白鼠的保护作用不低于Ｔｙ２─Ｖｉ。被动保护试验证明,在同等条件下,两种Ｖｉ抗原的免疫血清对小白鼠提供的保护作用相同。     

伤寒─鼠伤寒重组株Vi4072在小鼠中定居及血清学效果观察试验

以伤寒─鼠伤寒双价重组株Ｖｉ４０７２的３×１０８ＣＦＵ一次口服感染ＢＡＬＢ／Ｃ小鼠,４天后即可从小鼠的集合淋巴结、肝、脾中分离到该菌,４９天后该菌始被小鼠机体彻底清除。血清和小肠匀浆液中Ｖｉ抗体检测结果证明Ｖｉ４０７２菌株有刺激特异性免疫应答的功能。血清、小肠匀浆液中Ｖｉ抗体明显升高。