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。     

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。     

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。     

甲型副伤寒菌O—特异性多糖与破伤风类毒素结合菌苗的制备 …

以脱毒后去除类脂Ａ的甲型副伤寒杆菌高分子量Ｏ－ＳＰ１和低分子量Ｏ－ＳＰ２为特异笥抗原,以破伤风类毒素（ＴＴ）为蛋白质载体,用已二酸二肼（ＡＤＨ）作为连接剂制备的两种结合物及其多糖免疫ＮＩＨ小鼠,结果显示单独注射Ｏ＿ＳＰ１或Ｏ－ＳＰ２免疫小鼠后,均不能刺激小鼠产生抗ＬＰＳ抗体;而用Ｏ－ＳＰ１－ＴＴ和Ｏ－ＳＰ２－ＴＴ结合物免疫后,小鼠血甭中均产生了特异性抗－ＬＰＳ－ＩｇＧ抗体,且Ｏ－ＳＰ１－ＴＴ免疫组     

宋内氏痢疾杆菌O-SP-TT结合疫苗的制备及其免疫学特性的研究

宋内氏痢疾杆菌（S.Sonnei)的一个重要保护性抗原是菌体抗原（O抗原）即细菌细胞壁中的脂多糖（LPS）成分,已证实血清中足够水平的抗LPS IgG抗体抗体即能对该菌提供免疫保护作用。本文选用Westphal热酚法提纯宋内氏痢疾菌LPS,经酸水解法进一步脱毒处理后得到无毒但却具弱免疫原性的O-特异性多糖（O－SP）,然后采用化学方法即通过连接剂己二酸二肼（ADH）将其共价结合到破伤风类毒素（TT）上,共制备得到了三批宋内氏痢疾杆菌O－SP－TT结合疫苗。经生化和免疫学方法检测证实我们提纯的LPOS,OSP及其合成的L－SP－AH衍生物,O－SP－TT结合物均具有宋内氏痢疾杆菌O抗原特异性,同时其核酸和杂蛋白质含量低（≤2%),表明纯度较高。同时经小鼠免疫原性试验证实三批结合物免疫小鼠后产生的抗LPS IgG抗体滴度均比单一O－SP免疫后产生的要高出10倍以上,且结合物再次注射后存在加强应答。补体介导的体外杀菌力试验表明结合物免疫小鼠后诱导产生的血清抗LPSIgG抗体对宋内氏痢疾杆菌具特异性杀菌活性。本文结果表明宋内氏痢疾杆菌O-SP-TT结合疫苗可望作为一种有效的侯选痢疾疫苗做进一步的大规模人体观察。     

铜绿假单胞菌6型O-特异性多糖与破伤风类毒素结合物免疫原性研究

目的研制有效防治铜绿假单胞菌感染的疫苗。方法用热酚水法提取铜绿假单胞菌6型(IATS 6)菌株的脂多糖(LPS),去除类脂A并纯化O-特异性多糖(O-SP),用CDAP活化O-SP,己二酸二肼作连接臂,在EDAC作用下,与破伤风类毒素结合制备出O-SP-TT结合物并对该结合物进行小鼠免疫原性试验和免疫保护性试验。结果制备的结合物在小鼠免疫原性试验中,产生了高效价的IgG抗体,与O-SP试验组相比,差异具有统计学意义(P<0.05);免疫保护性试验表明,结合物免疫小鼠能很好的保护5～10 LD50活菌的腹腔攻击。结论该结合物有望成为防治IATS 6型铜绿假单胞菌感染的有效疫苗。     

6A型肺炎链球菌荚膜多糖水解物的制备及其结合物在小鼠体内免疫原性研究

目的制备6A型肺炎链球菌荚膜多糖(PS6A)水解物及其结合物,研究结合物在小鼠体内的免疫原性。方法在60℃水解温度下,筛选出PS6A水解适宜的醋酸水解浓度和水解时间。通过水解物和原糖(PS6A)的核磁共振氢谱(1H NMR)和磷谱(31P NMR)比较验证水解物结构的正确性。以1-氰基-4-二甲胺基吡啶四氟硼酸盐(1-Cyano-4-(dimethylamino)pyridinium tetrafluoroborate,CDAP)活化水解物的羟基,并与破伤风类毒素己二酸酰肼衍生物(TTAH)结合,制备结合物PS6A-TTAH;并检测结合物的理化性质。通过抑制ELISA分析原糖、水解物和结合物的抗原性。将小鼠分为2组(PS6A组和PS6A-TTAH组),分别免疫3剂次,用间接ELISA分析原糖和结合物在小鼠体内的免疫原性。结果多糖经0.2 mol/L醋酸60℃水解8 h后,其相对分子质量大小(KD)由0.03升高为0.51,重均相对分子质量(Mw)由8.127×10~5g/mol降为1.175×10~5g/mol。核磁共振结果表明,各特征质子的化学位移相比原糖未发生改变。结合物的游离多糖质量分数为4.55%,游离载体蛋白质质量分数为1.08%。抑制ELISA结果显示,水解物、结合物的抑制曲线与原糖基本吻合。PS6A-TTAH组有剂次加强效应(P分别为0.001、0.004和0.001),其第1~3针免后IgG抗体水平均高于PS6A组,差异均具有统计学意义(P分别为0.001、0.002和0.001)。结论制备的PS6A水解物能有效的保留天然多糖的特异基团,以此水解物制备的结合物在小鼠体内具有良好的免疫应答。     

B群链球菌Ia型多糖─破伤风类毒素结合菌苗对抗Ia、Ib型保护性抗体产生的刺激作用

Ｂ群链球菌Ｉａ型多糖─破伤风类毒素结合菌苗对抗Ｉａ、Ｉｂ型保护性抗体产生的刺激作用作者将Ｂ群链球菌（ＢＧＳ）Ｉａ型菌株的荚膜多糖侧链上的唾液酸氧化后,通过还原氨化反应,同破伤风类毒素（ＴＴ）结合,制备成含１２％蛋白、８８％多糖的Ｉａ－ＴＴ结合菌苗。分...     

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。     

伤寒Vi-rEPA结合疫苗在小鼠中的免疫原性研究

以伤寒Vi多糖疫苗及结合物含量分别为20％、40％、60％的伤寒Vi-rEPA结合疫苗经皮下免疫小鼠后眼眶采血分离血清,用ELISA法测血清抗体浓度。血清抗体分析表明,伤寒Vi多糖疫苗和伤寒Vi-rEPA结合疫苗均有良好的免疫原性,但伤寒Vi-rEPA结合疫苗的免疫原性优于伤寒Vi多糖疫苗;伤寒Vi多糖疫苗无加强免疫应答效应,而伤寒Vi-rEPA结合疫苗有加强免疫应答效应;不同高分子结合物含量的伤寒Vi-rEPA结合疫苗免疫小鼠,其结合物含量为40％、60％的伤寒Vi-rEPA结合疫苗比结合物含量为20％的结合疫苗具有更好的免疫原性,而前二者的免疫原性无显著性差异。     

An oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus

We have developed an oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus. Purified group B streptococcal type III capsular polysaccharide was depolymerized by enzymatic digestion using endo-beta-galactosidase produced by Citrobacter freundii. Following enzymatic digestion, oligosaccharides were fractionated by gel filtration chromatography on Sephadex G-75. An oligosaccharide pool of average Mr = 14,500 (corresponding to 13.6 repeating units of the type III polysaccharide) was used for conjugation to tetanus toxoid. Tetanus toxoid was covalently coupled via a synthetic spacer molecule to the reducing end of the oligosaccharide by reductive amination. The oligosaccharide-tetanus toxoid conjugate elicited type III-specific anticapsular antibodies (measured in enzyme-linked immunosorbent assay) in three out of three rabbits whereas the unconjugated native type III polysaccharide was nonimmunogenic. Antiserum from rabbits vaccinated with the oligosaccharide-protein conjugate protected mice against lethal challenge with live group B streptococci (16 out of 16 mice survived) and opsonized group B streptococci for phagocytosis in vitro. No protection was conferred by preimmune serum nor by serum from rabbits vaccinated with unconjugated native type III polysaccharide. An oligosaccharide-protein conjugate vaccine of this design may prove to be an effective immunogen for protection against group B streptococcal infection in humans. In addition, the approach to vaccine design utilized in these studies will facilitate further definition of the structural parameters that determine immune response to glycoconjugate vaccines.     

破伤风类毒素抗体酶联双抗原夹心法定量检测试剂的研制及评价

研制破伤风类毒素抗体酶联双抗原夹心法定量检测试剂,用于破伤风免疫血浆抗体效价检测。以精制破伤风类毒素经Sephacryl S-300柱层析纯化后作为包被抗原,用辣根过氧化物酶以改良过碘酸钠法标记精制破伤风类毒素作为酶标记抗原,以破伤风人免疫球蛋白国家标准品采用小鼠中和试验法标定试剂盒定量标准品,制备双抗原夹心法定量检测试剂;进行试剂盒检测范围、特异性、重复性、精密度及稳定性考核,并与小鼠中和试验法、琼脂双扩散法及国外破伤风类毒素抗体酶联试剂盒进行比较。结果显示,试剂盒的检测范围为10~150mIU/ml,灵敏度为10mIU/ml,线性好(r>0.996),板内孔间变异度小(CV<8%),特异性强(100%),重复性好(CV<13%),于37℃放置6天测定结果无明显差异,与小鼠中和试验法、英国Biding Site酶联试剂有良好的一致性。试验证明所研制的试剂盒适用于破伤风免疫血浆中的破伤风抗体效价定量检测。     

Vaccination response to tetanus toxoid and 23-valent pneumococcal vaccines following administration of a single dose of abatacept: a randomized,open-label,parallel group study in healthy subjects

The effect of abatacept, a selective T-cell co-stimulation modulator, on vaccination has not been previously investigated. In this open-label, single-dose, randomized, parallel-group, controlled study, the effect of a single 750 mg infusion of abatacept on the antibody response to the intramuscular tetanus toxoid vaccine (primarily a memory response to a T-cell-dependent peptide antigen) and the intramuscular 23-valent pneumococcal vaccine (a less T-cell-dependent response to a polysaccharide antigen) was measured in 80 normal healthy volunteers. Subjects were uniformly randomized to receive one of four treatments: Group A (control group), subjects received vaccines on day 1 only; Group B, subjects received vaccines 2 weeks before abatacept; Group C, subjects received vaccines 2 weeks after abatacept; and Group D, subjects received vaccines 8 weeks after abatacept. Anti-tetanus and anti-pneumococcal (Danish serotypes 2, 6B, 8, 9V, 14, 19F and 23F) antibody titers were measured 14 and 28 days after vaccination. While there were no statistically significant differences between the dosing groups, geometric mean titers following tetanus or pneumococcal vaccination were generally lower in subjects who were vaccinated 2 weeks after receiving abatacept, compared with control subjects. A positive response (defined as a twofold increase in antibody titer from baseline) to tetanus vaccination at 28 days was seen, however, in ≥ 60% of subjects across all treatment groups versus 75% of control subjects. Similarly, over 70% of abatacept-treated subjects versus all control subjects (100%) responded to at least three pneumococcal serotypes, and approximately 25–30% of abatacept-treated subjects versus 45% of control subjects responded to at least six serotypes.     

双佐剂包被的破伤风类毒素马匹免疫试验

为提高破伤风免疫马匹的血浆抗体效价,应用不同佐剂配制TT抗原,进行马匹超免疫比较研究;采用FIA和植物油双佐剂包被与单佐剂包被的TT抗原,注射马匹进行超免疫,比较三组血浆的效价;结果显示,双佐剂抗原较单佐剂的免疫效果好,但可能对马匹刺激较强,有待调整注射剂量和免疫程序。     

Induction of meningococcal group B polysaccharide-specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine

Conjugation of the group B meningococcal polysaccharide to tetanus toxoid failed to substantially enhance its immunogenicity in mice. Therefore, additional chemical manipulation of the basic structure of the group B meningococcal polysaccharide was attempted, on the premise that a synthetically derived artificial antigen might be capable of modulating the immune response in mice to produce elevated levels of cross-reactive group B meningococcal polysaccharide-specific antibodies. To achieve this, the antigenicity of the modified polysaccharide to group B meningococcal polysaccharide-specific antibodies had to be preserved, and this criterion could only be satisfied in modifications in which the carboxylate and N-carbonyl groups of the sialic acid residues of polysaccharide remained intact. Therefore, the most successful modifications were accomplished by N-deacetylation of the group B meningococcal polysaccharide with strong base to yield a precursor that could then be N-acetylated or N-arylated with different substituents. For example, the introduction of N-propionyl groups, followed by conjugation of the resultant N-propionylated group B meningococcal polysaccharide to tetanus toxoid, yielded an antigen that when injected in mice induced in them high levels of cross-reactive group B meningococcal polysaccharide-specific IgG antibodies. The T cell dependency of this antigen was established when it was demonstrated that the levels of these B polysaccharide-specific antibodies could be significantly boosted by using both the N-propionylated- and native N-acetylated-group B meningococcal polysaccharide-tetanus toxoid conjugates.     

In vitro human antibody production to the Haemophilus influenzae type b capsular polysaccharide

In vitro production of human antibody to the Haemophilus influenzae type b capsular polysaccharide (PRP) and to tetanus toxoid (TT) and diphtheria toxoid was measured in culture supernatants of peripheral blood mononuclear cells and by enumeration of antibody secreting cells (AbSC) in an enzyme-linked immunosorbent-plaquing assay. Normal adult peripheral blood mononuclear cells stimulated with Epstein-Barr virus secreted anti-PRP antibody with a frequency of 1/552 to 1/1190 relative to total Ig secreting cells; the frequency of AbSC to tetanus toxoid (TT) was 7.5 times higher (p less than 0.05). These frequencies did not change significantly after in vivo immunization, although the isotype distribution shifted toward increased IgG for TT and increased IgG and IgA for PRP. At 8 days postimmunization, spontaneous AbSC to PRP and TT were detected; frequencies for total anti-TT AbSC again being higher than anti-PRP, but there were significantly more IgA plaques among anti-PRP AbSC. Spontaneous AbSC were suppressed in culture by pokeweed mitogen and enhanced by cyclosporine. Three wk after in vivo immunization with PRP and TT, in vitro stimulation with pokeweed mitogen, Staphylococcus aureus Cowan 1 bacteria, or antigen induced anti-TT but not anti-PRP in vitro antibody secretion, although Epstein-Barr virus induced both. These data suggest that PRP, a polysaccharide, and TT, a protein, differ in their requirements for in vitro activation with antigen and mitogens.     

Functional properties of human B cell subpopulations defined by monoclonal antibodies HB4 and FMC7

Human B cell subpopulations can be distinguished by the expression of B cell-associated antigens. mAb directed against these structures allow the isolation and subsequent functional analysis of such subsets. Blood B cells from healthy individuals were separated on basis of the expression of the antigens recognized by the mAb HB4 and FMC7. The B cells present in the thus obtained populations were analyzed for their ability to secrete IgM and IgG after stimulation in vitro with polyclonal B cell activators (PWM, Staphylococcus aureus Cowan I), antigens (tetanus toxoid, type 4 pneumococcal polysaccharides), and soluble B cell differentiation factors. The results suggest that B cells capable of Ig and anti-tetanus toxoid or anti-type 4 pneumococcal polysaccharide antibody production after in vitro culture are localized in a relative small B cell subpopulation carrying the FMC7 determinant but lacking HB4. This holds true for both the IgM- and IgG-secreting B cells. These data further suggest that the majority of B cells found in the blood and which can be characterized as being surface IgM+/IgD+ HB4+ are immature cells unable to respond to differentiation-inducing signals.     

Conjugation of tetanus toxoid with Salmonella typhimurium PTCC 1735 O-specific polysaccharide and its effects on production of opsonizing antibodies in a mouse model

Serious enteric and extra-intestinal infections with Salmonella typhimurium are very common in many human populations. Phagocytosis is the main defense mechanism against this bacterium; however, the unique structure of S. typhimurium lipopolysaccharide (LPS) makes it resistant to opsonization by complement components. In the present study, the S. typhimurium LPS O-chain was purified and conjugated with tetanus toxoid and the effects of the conjugated vaccine (O-specific polysaccharide tetanus toxoid (O-SP-TT)) on induction of specific antibodies were investigated in a mouse model. In vitro assays measuring phagocytosis in the presence of opsonizing antibodies were performed. Three subcutaneous injections of the O-SP-TT conjugate conferred protection against the intraperitoneal challenge with S. typhimurium and the LD50 was greater for immunized animals than for controls. The mean number of ingested S. typhimirium / mouse peritoneal cell in the presence of sera obtained from immunized mice with purified O-chain, O-SP-TT conjugate, heat-killed bacteria, and negative control were 6.96, 14.24, 15.96, and 6.67, respectively. In summary, we developed an O-SP-TT conjugate that induced opsonizing antibodies that increased phagocytosis, as determined by in vitro assays. In addition, chemiluminescence results, an indicator of oxidative burst, indicated that peritoneal cells respond better to live S. typhimurium cells in the presence of sera obtained from O-SP-TT conjugate immunized mice.