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。     

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.     

A群C群脑膜炎球菌结合疫苗稳定性

目的评价A群C群脑膜炎球菌结合疫苗原液和成品的稳定性。方法分别将A群、C群脑膜炎球菌结合疫苗原液及A群C群脑膜炎球菌结合疫苗各选取连续3批,分别放置于37℃、20~25℃和2~8℃3种温度下,在一定的时间取样进行主要项目测定,在关键时间点进行全面检测。结果 A群结合疫苗原液于2~8℃保存9个月,20~25℃保存4周,37℃保存4 d;C群结合疫苗原液于2~8℃保存9个月,20~25℃保存6个月,37℃保存4周;A群C群脑膜炎球菌结合疫苗于2~8℃保存2年3个月,20~25℃保存6个月,37℃可以保存9周;各项检测指标均符合质量标准的要求。结论在2~8℃条件下,A群、C群脑膜炎球菌结合疫苗原液存放6个月,A群C群脑膜炎球菌结合疫苗存放2年,其质量稳定。     

Immunoprotective potential of polysaccharide-tetanus toxoid conjugate in Klebsiella pneumoniae induced lobar pneumonia in rats

The polysaccharide (PS) derived from K. pneumoniae NCTC 5055 lipopolysaccharide (LPS) was covalently linked to tetanus toxoid by using carbodimide with adipic acid dihydrazide as a spacer molecule. The conjugate was found to be non-toxic and non-pyrogenic at 100 microg dose level. At a similar dose, the conjugate did not elicit any local skin reaction on intradermal preparatory injection in rabbits. The conjugate was immunoprotective as was evident from the decrease in relative colonization of bacteria in lungs of immunized rats as compared to the control animals. Immunization with the conjugate resulted in alveolar macrophage activation in terms of their ability to phagocytose bacteria in vitro.     

W135群脑膜炎球菌结合物小鼠免疫血清IgG抗体亚类和亲合力的检测

目的通过检测W135群脑膜炎球菌结合物(简称W135群结合物)小鼠免疫血清中IgG抗体亚类及IgG抗体亲合力,判断W135群结合物的免疫应答特征。方法 (1)免疫血清的制备:将NIH雌性小鼠随机分为3组(A组、B组、对照组),每组30只。A组采用含2.5μg多糖的PSW135CNBr-TT分别于0、2、4、8周腹股沟皮下注射小鼠;B组采用含2.5μg多糖的PSW135CNBr-TT分别于0、4、8周腹股沟皮下注射小鼠;对照组采用含2.5μg多糖的PSW135分别于0、2、4周腹股沟皮下注射小鼠。分别于每次免疫7 d后,经小鼠眼眶静脉采血,分离血清备用;(2)IgG抗体亚类和亲合力的检测:采用间接ELISA分析各组免疫血清中IgG抗体及IgG抗体亚类含量;采用单一浓度的硫氰酸钾溶液作为洗脱液,用间接ELISA测定免疫血清中IgG相对亲合力。结果 A组和B组获得的小鼠免疫血清,IgG抗体均以IgG1为主(占总IgG抗体的80%以上),IgG抗体及其亚类抗体含量均呈典型的剂次加强效应;对照组各针次免疫血清中的IgG3抗体含量所占比例最高,未见免疫剂次加强效应。A组IgG抗体的相对亲合力分别是46.6%、25.4%、17.6%和47.6%,B组IgG抗体的相对亲合力分别是46.6%、23.6%和48.3%,对照组IgG抗体的相对亲合力分别是92.4%、81.9%和81.0%。结论 W135群结合物小鼠免疫血清呈现典型的胸腺依赖性(thymus dependent,TD)抗原的免疫应答特征。     

不同稀释液对冻干A、C群脑膜炎球菌结合疫苗质量的影响

为选择合适的的冻干A、C群脑膜炎球菌结合疫苗的稀释液,以注射用水(H2O)、生理盐水(NS)、磷酸盐缓冲液(PBSpH6.8～7.2)作为候选稀释液,进行了相关溶解度、pH、渗透压、异常毒性、免疫原性的检测比较。溶解度、pH、免疫原性结果显示三种稀释液无差异;H2O作为稀释液的渗透压不符合人用制剂渗透压的要求,另两者则符合要求;三种稀释液溶解制品后的pH检测数据均符合要求,但PBS的缓冲能力更强。故认为PBS更适合作为冻干A、C群脑膜炎球菌结合疫苗的稀释液。     

Down-regulation of IgE and IgG4 antibodies to tetanus toxoid and diphtheria toxoid by covaccination with cellular Bordetella pertussis vaccine

Pertussis (P) toxin acts as adjuvant for IgE formation against simultaneously administered Ags in animal models. P vaccination may also have an adjuvant impact on IgE formation against coadministered diphtheria (D) and tetanus (T) Ags in humans. Sera of 103 D-T-P-immunized and 319 D-T-immunized children aged 2 years were analyzed for IgE, IgG4, and IgG to D and T (radioallergosorbent test), total IgE and IgE against common inhalant allergens (CAP radioallergosorbent test fluoroenzyme immunoassay). Fewer D-T-P- than D-T-immunized children had sera positive for T-IgE (12.6 vs 53.6%, p < 0.001), T-IgG4 (71.6 vs 89.2%, p < 0.001), D-IgE (31.0 vs 70.5%, p < 0.001), and D-IgG4 (85.2 vs 93.4%, p = 0.039). Suppression of T-IgE was not dependent on the cutoff chosen for a positive test result, but was dependent on the proportion of D-T immunizations given with P. The risk for sensitization to common environmental allergens did not differ (odds ratio 0.953, 95% confidence interval 0.815-1.114). No significant differences between D-T- and D-T-P-immunized children were found with regard to T-IgG or D-IgG. In summary, IgE and IgG4 (but not IgG) serum levels to coadministered D- and T-Ags are suppressed among P-immunized children as compared with nonimmunized children. These results suggest that the presence of a microbial product during Ag exposure can down-regulate an IgE/IgG4 response in humans.     

Correlation of group C meningococcal conjugate vaccine response with B- and T-lymphocyte activity

Despite the success of conjugate vaccination against meningococcal group C (MenC) disease, post-vaccination, some individuals still exhibit rapid waning of initially protective bactericidal antibody levels. The mechanism of this relative loss of humoral protection remains undetermined. In this report we have investigated the relationship between T- and B-cell activation and co-stimulation and the loss of protective antibody titers. We have found that healthy volunteers who lose protective MenC antibody levels one year after receipt of glycoconjugate vaccine exhibit no detectable cellular defect in polyclonal B- or T-cell activation, proliferation or the B-memory pool. This suggests that the processes underlying the more rapid loss of antibody levels are independent of defects in either initial T- or B-cell activation.     

C群奈瑟氏脑膜炎球菌荚膜多糖-TT结合疫苗的制备及其在小鼠体内免疫原性的研究

将C群脑膜炎球菌荚膜多糖以ADH作为间隔剂与TT结合形成GCMP-TT结合疫苗,然后用此结合疫苗免疫NIH小鼠,结果显示使用GCMP免疫小鼠后仅能产生较低水平抗GCMP的IgG抗体,而用GCMP-TT免疫后小鼠血清中产生了较GCMP免疫显著增高抗GCMP的IgG抗体,并且GCMP-TT组第二次和第三次免疫后与初次免疫相比,IgG抗体水平均有显著升高(P<0.01),表明GCMP-TT结合疫苗具有免疫记忆和再次免疫加强应答效应。补体介导的血清抗体体外杀菌试验结果证明,GCMP-TT结合疫苗组免疫小鼠诱导的抗体IgG比GCMP组具有增强的体外杀菌活性。     

速率比浊法检测ACYW135群脑膜炎球菌结合疫苗中各群多糖含量

目的建立速率比浊法准确检测ACYW135群脑膜炎球菌结合疫苗(group ACYW135 meningococcal conjugate vaccine)的各群多糖含量。方法制备多糖参考品和抗血清,建立检测ACYW135群脑膜炎球菌结合疫苗各群多糖含量的速率比浊法,对建立的方法进行验证及初步应用。结果①建立的方法具备高度的特异性,各群血清仅与本群多糖抗原发生特异性反应,与非本群多糖抗原无交叉反应;②在质量浓度为0.5～5.0μg/mL多糖参考品检测区间线性良好,相关系数r>0.98;③各群血清的最低检测限均低于质量浓度为0.35μg/mL;④批内和批间变异系数(CV)值分别为0.83%～5.34%和2.33%～13.88%,加样回收率为90.5%～118.4%,精密度和准确度均符合常规质量控制要求;⑤初步应用结果显示,建立的方法可准确检测疫苗的各群多糖含量,且与火箭电泳法检测结果差异无统计学意义(P>0.05)。结论建立的方法简便快捷、灵敏准确、自动化程度高,可有效检测ACYW135群脑膜炎球菌结合疫苗的各群多糖含量。     

B cell memory to a serogroup C meningococcal conjugate vaccine in childhood and response to booster: little association with serum IgG antibody

The maintenance of adequate serum Ab levels following immunization has been identified as the most important mechanism for individual long-term protection against rapidly invading encapsulated bacteria. The mechanisms for maintaining adequate serum Ab levels and the relationship between Ag-specific memory B cells and Ab at steady state are poorly understood. We measured the frequency of circulating serogroup C meningococcal (MenC)-specific memory B cells in 250 healthy 6- to 12-y-old children 6 y following MenC conjugate vaccine priming, before a booster of a combined Haemophilus influenzae type b-MenC conjugate vaccine and then 1 wk, 1 mo, and 1 y after the booster. We investigated the relationship between circulating MenC-specific memory B cell frequencies and Ab at baseline and following the booster vaccine. We found very low frequencies of circulating MenC-specific memory B cells at steady state in primary school-aged children and little association with MenC IgG Ab levels. Following vaccination, there were robust memory B cell booster responses that, unlike Ab levels, were not dependent on age at priming with MenC. Measurement of B cell memory in peripheral blood does not predict steady state Ab levels nor the capacity to respond to a booster dose of MenC Ag.     

Structurally identical capsular polysaccharide expressed by intact group B streptococcus versus Streptococcus pneumoniae elicits distinct murine polysaccharide-specific IgG responses in vivo

We previously reported distinct differences in the murine in vivo Ig polysaccharide (PS)-specific responses to intact Streptococcus pneumoniae compared with responses to Neisseria meningitidis and that in each case, the bacterial subcapsular domain markedly influences the Ig response to the associated PS. In light of potentially unique contributions of biochemically distinct capsular PS and/or their characteristic attachments to the underlying bacterium, it remains unresolved whether different bacterial subcapsular domains can exert differential effects on PS-specific Ig responses to distinct bacterial pathogens. In this report, we used a mutant strain of group B Streptococcus (Streptococcus agalactiae) type III (GBS-III) that expresses desialylated capsular polysaccharide of GBS-III, biochemically identical to capsular pneumococcal polysaccharide type 14 (PPS14) of Streptococcus pneumoniae (intact inactivated Streptococcus pneumoniae, capsular type 14, Pn14), directly to compare the in vivo PPS14-specific IgG responses to two distinct gram-positive bacteria. Although both GBS-III and Pn14 elicited relatively rapid primary PPS14-specific IgG responses dependent on CD4(+) T cells, B7-dependent costimulation, and CD40-CD40L interactions, only GBS-III induced a highly boosted ICOS-dependent PPS14-specific IgG response after secondary immunization. Of note, priming with Pn14 and boosting with GBS-III, although not isolated PPS14, elicited a similar boosted PPS14-specific IgG response that was dependent on CD4(+) T cells during secondary immunization, indicating that Pn14 primes for memory but, unlike GBS-III, fails to elicit it. The inability of Pn14 to elicit a boosted PPS14-specific IgG response was overcome by coimmunization with unencapsulated GBS-III. Collectively, these data establish that structurally identical capsular PS expressed by two distinct gram-positive extracellular bacteria can indeed elicit distinct PS-specific IgG responses in vivo.     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Protective efficacy of maternal antibodies induced by Salmonella toxoid (vaccine)

An attempt was made to evaluate the protective efficacy of maternal antibodies in chicks against salmonellosis. Layer chicks ageing 21 days were individually vaccinated with 100 microg of Salmonella enterica subspecies enterica serovar Weltevreden (BM 1643) toxoid adjuvanted with vitamin E subcutaneously. After 90 days of the primary vaccination the birds were given booster dose of the vaccine. The saline extract of the yolk of eggs laid by the vaccinated birds yielded agglutination and ELISA titres ranging from 43.2 +/- 5.33 to 75.2 +/- 6.26 and 4.987 x 10(3) +/- 0.54 to 5.89x103 +/- 0.56, respectively. Sera of chicks hatched from eggs laid by the vaccinated layers were also subjected to agglutination and ELISA. Agglutination and ELISA titres on the 5th day--post hatching (dph) were 21.6 +/- 1.75 and 4.025 x 10(2) +/- 0.59, while on the 10th dph titers were 13.6 +/- 1.65 and 1.21 x 10(2) +/- 0.60, respectively. It was also observed that only one out of 6 chicks died when challenged with 2 x 10(9) CFU of S. serovar Gallirarum at the age of 7 days showing 83.33% protection. Thus it can be concluded that passive immunity confided by Salmonella enterica subspecies enterica serovar Weltevreden (BM 1643) toxoid can protect chicks against salmonellosis during their early days of life.     

Intranasal administration of a Schistosoma mansoni glutathione S-transferase-cholera toxoid conjugate vaccine evokes antiparasitic and antipathological immunity in mice.

Mucosal administration of Ags linked to cholera toxin B subunit (CTB) can induce both strong mucosal secretory IgA immune responses and peripheral T cell hyporeactivity. In this study, intranasal (i.n. ) administration of CTB-conjugated Schistosoma mansoni 28-kDa GST (CTB-Sm28GST) was found to protect infected animals from schistosomiasis, especially from immunopathological complications associated with chronic inflammation. Worm burden and liver egg counts were reduced in infected animals treated with the CTB-Sm28GST conjugate as compared with mice infected only, or with mice treated with a control (CTB-OVA) conjugate. However, a more striking and consistent effect was that granuloma formations in liver and lungs of mice treated with CTB-Sm28GST were markedly suppressed. Such treatment was associated with reduced systemic delayed-type hypersensitivity and lymphocyte proliferative responses to Sm28GST. Production of IFN-gamma, IL-3, and IL-5 by liver cells was also markedly reduced after i.n. treatment of CTB-Sm28GST, whereas IL-4 production was not impaired. Intranasal treatment of infected mice with CTB-Sm28GST increased IgG1-, IgG2a-, IgA-, and IgE-Ab-forming cell responses in liver in comparison with treatment with CTB-OVA, or free Sm28GST. Most importantly, mucosal treatment with CTB-Sm28GST significantly reduced animal mortality when administered to chronically infected mice. Our results suggest that it may be possible to design a therapeutic vaccine against schistosomiasis that both limits infection and suppresses parasite-induced pathology.     

Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity-purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity-purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.     

Production of anti-sporozoite antibodies in absence of response to carrier by coupling an MDP derivative to a malaria peptide-tetanus toxoid conjugate

A synthetic peptide (pep) representing a portion of the Plasmodium knowlesi circumsporozoite protein attached to a tetanus toxoid (TT) carrier, has been shown to be immunogenic when delivered in saline with derivatives of the synthetic adjuvant, muramyl dipeptide (MDP). The present study was designed to determine if the degree of substitution of pep and of MDP derivatives on the tetanus toxoid (TT) carrier, as well as the choice of MDP derivative used play a role in determining anti-pep and anti-TT antibody levels. One of the MDP derivatives used in the conjugates was epsilon-amino-caproic Murabutide, since Murabutide which is currently in clinical trials cannot be conjugated. The results show that low doses of this derivative coupled with pep on TT can be used to stimulate high levels of circulating anti-pep antibodies without augmenting the anti-carrier response. In addition, anti-pep antibodies elicited in response to one of the conjugates were biologically active since they produced shedding of the circumsporozoite coat of live parasites.     

C群流脑多糖菌苗唾液酸含量测定试验

Ｃ群脑膜炎球菌多糖菌苗是我们近年开发的一种新制品,其中唾液酸（ＳＡ）是该制品的有效成份之一。为了严格控制该制品的质量,经反复试验,对比各种试验条件,选择出较理想的测定ＳＡ方法－改良法,该法与Ｓｖｅｎｎｅｒｈｏｌｍ氏法比较,其标准曲线变异系数（ｃｖ％）为０．０３％,低于Ｓｖｅｎｎｅｒｈｏｌｍ氏法（０．３１％）;其回收率平均为１００．６２％。与中检所方法比较,无显著差异（Ｐ〉０．０５）,对批样品进行６