Isoelectric focusing of human antibody to the Haemophilus influenzae b capsular polysaccharide: restricted and identical spectrotypes in adults

Serum antibody to the capsular polysaccharide of Haemophilus influenzae b of human adults was analyzed by isoelectric focusing. Restricted antibody spectrotype patterns were commonly observed with as few as one spectrotype in some subjects after immunization with the isolated capsular polysaccharide. Some patterns were as restricted as human hybridoma antibody. There was no correlation of antibody titer and heterogeneity of patterns. The dominant spectrotype persisted unchanged for over 2 yr after immunization, and the pattern detected in preimmunization serum samples persisted unchanged after immunization. Indistinguishable patterns were commonly observed in genetically unrelated adults. Adults immunized with conjugate vaccines, which were composed of oligosaccharides prepared from the capsular polysaccharide that were covalently linked to protein carriers, also produced restricted serum antibody spectrotype patterns. Immunization with the cross-reactive polysaccharide of E. coli K100 induced a spectrotype pattern that was restricted but different from that induced by the H. influenzae b capsular polysaccharide.     

Specific characteristics of composition of synthetic nutrient media for cultivation of Hemophilus influenzae type b from the standpoint of bacterial metabolism

In many countries vaccination against Haemophilus influenzae of type b (Hib) has permitted the liquidation of severe generalized forms of infections caused by these bacteria. The vaccine is obtained on the basis of Hib capsular polysaccharide. To obtain pure capsular polysaccharide, Hib should be cultivated on synthetic nutrient media. The present review deals with the data substantiating the advantages of using synthetic nutrient media for the cultivation of these bacteria with a view to obtaining pure capsular polysaccharide.     

Influence of the aminopeptide concentration on the growth of Haemophilus influenzae, type b, and the synthesis of its capsular polysaccharide

The influence of the aminopeptide concentration on the growth of H. influenzae b culture and the synthesis of H. influenzae b capsular polysaccharide was determined. The maximum amount of capsular polysaccharide was accumulated at the concentration of aminopeptide in the culture fluid reaching 50 ml/l. An increase in the aminopeptide concentration led to a decreased amount of synthesized polysaccharide and an increased amount of biomass. The decrease of the aminopeptide concentration to 10 ml/l resulted in decreased amounts of both biomass and synthesized polysaccharide.     

Determination of the structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with the capsule from type b Haemophilus influenzae

The structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with that from type b Haemophilus influenzae, was determined by using a combination of chemical and spectroscopic techniques. The structure of the K100 repeating unit was found to be----3)-beta-D-Ribf-(1----2)-D-ribitol-5-(PO4----. The K100 polysaccharide is thus identical in composition to, but different in linkage from, the H. influenzae type b capsular polysaccharide, which has beta-D-Ribf-(1----1)-D-ribitol linkages.     

Influence of nicotinamide adenine dinucleotide and hemin concentrations on the growth of Haemophilus influanzae type b and the synthesis of capsular polysaccharide

In the process the cultivation of H. influenzae, type b, in semisynthetic nutrient medium with aminopeptide base the growth of the bacteria and the synthesis of capsular polysaccharide were shown to depend on the concentrations of aminopeptide, nicotinamide adenine nucleotide (NAD) and hemin. An increase in the concentrations of NAD and hemin stimulated the growth of H. influenzae and inhibited the synthesis of capsular polysaccharide. Similar effect was observed in the simultaneous increase of NAD and hemin concentrations. At elevated concentrations of NAD and hemin and the content of aminopeptide equal to 350 mI/l the maximum weight of biomass was achieved. The increase of hemin concentration had no influence on the growth of H. influenzae, type b, and the synthesis of capsular polysaccharide.     

Haemophilus influenzae type b polysaccharide-protein conjugate vaccine

An Haemophilus influenzae type b capsular polysaccharide-protein conjugate has been prepared. The polysaccharide was coupled to the serotype II protein of group B meningococcus through the spacer 6-aminocaproic acid using cyanogen bromide and water soluble carbodiimide. The conjugate can be shown to be reproducible and is stable and highly immunogenic in mice and African green monkeys. Clinical evaluation of this conjugate in children 3 months to 4 years of age showed that it elicited an antibody titer to the polysaccharide moiety greater than 1000 ng/ml in children 8 months of age or older.     

一种新的CTAB加入方法对A群脑膜炎球菌荚膜多糖分子大小的影响

目的探讨CTAB不同的加入方法对A群脑膜炎球菌荚膜多糖分子大小的影响。方法采用分次加入手动搅拌和持续加入机械快速搅拌两种CTAB加入方法,纯化获得荚膜多糖粗糖,分别编为B组和C组。将两组荚膜多糖粗糖分别纯化获得精糖,分别编为D组和E组。以Sepharose CL-4B凝胶层析纯化获得荚膜多糖并检测其KD值。结果 B组荚膜多糖粗糖的KD值介于0.34~0.35之间,C组荚膜多糖粗糖的KD值介于0.03~0.05,进一步用苯酚纯化获得精糖后KD值D组介于0.34~0.36之间,E组介于0.22~0.28之间。两组相比KD值显著降低。结论CTAB的加入过程对A群脑膜炎球菌荚膜多糖的分子大小有明显的影响,CTAB沉淀时进行快速而充分的搅拌,纯化获得的荚膜多糖相对分子质量更大。     

Effect of flavonoids on the composition of surface glycopolymers of Azospirillum lipoferum Sp59b

Cultivation of the type strain Azospirillum lipoferum Sp59b in the presence of flavonoid quercetin induced modification of the structure of the bacterial lipopolysaccharide. Cultivation in the presence of the flavonoid was shown to result in altered serological characteristics of the bacteria, increased heterogeneity of the outer membrane lipopolysaccharide pool, as well as in modified composition and fatty acid ratio of lipid A. The flavonoid was shown to induce the synthesis of the O-specific polysaccharide with the repeating structure represented by a tetrasaccharide consisting of a linear trisaccharide fragment of α-L-Rhap residues in the main chain and the terminal β-D-Glcp residue. The structure of this O-specific polysaccharide was identical to the previously determined structure of the capsular polysaccharide of these bacteria grown without quercetin. Modifications in the structural composition of the capsular polysaccharide induced by cultivation in the presence of quercetin were revealed.     

A群脑膜炎球菌荚膜多糖纯化工艺的优化

目的对A群脑膜炎球菌荚膜多糖纯化工艺的关键步骤进行分步研究,优化每一步工艺参数。方法优化十六烷基三甲基溴化铵的加入浓度、复合多糖的解离浓度和解离时间、不同厂家的苯酚、超滤和透析等工艺过程对荚膜多糖的影响。结果十六烷基三甲基溴化铵质量体积终浓度0.10%(w/v)沉淀效果更好,纯化获得的荚膜多糖产量更高相对分子质量更大。复合多糖解离浓度越高,纯化获得的荚膜多糖相对分子质量越小。延长复合多糖解离时间有利于提高荚膜多糖产量。不同厂家的苯酚、超滤和透析等工艺对荚膜多糖的产量和分子大小没有影响。结论现行A群脑膜炎球菌荚膜多糖纯化工艺复杂,优化后的工艺提高了荚膜多糖产量,缩短了工艺用时,增加了工艺稳定性。     

Structure of the capsular polysaccharide of Actinobacillus pleuropneumoniae serotype 5b.

The capsular polysaccharide of Actinobacillus pleuropneumoniae serotype 5b (strain L20) was found to be a high molecular mass polymer composed of 2-acetamido-2-deoxy-D-glucose, D-glucose, and 3-deoxy-D-manno-octulosonic acid (KDO). Methylation analysis, partial hydrolysis and a combination of homonuclear and 1H-detected heteronuclear shift-correlated nuclear magnetic resonance experiments showed the polysaccharide to be a branched polymer of a trisaccharide repeating unit, having the structure: [formula; see text]     

Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus

The capsular polysaccharide of group B Streptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with beta-N-acetylglucosaminidase, which catalyzes hydrolysis of the beta-D-GlcNAc(1-->4)beta-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and (31)P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.     

肺炎链球菌5型发酵工艺的优化

目的:采用正交试验设计方法进行肺炎链球菌5型发酵工艺的研究。方法:根据正交试验设计表L9(34)设计的试验条件组合进行了9次肺炎链球菌5型的发酵,采用70升发酵罐进行发酵工艺的摸索,提取了肺炎链球菌5型荚膜多糖粗糖。结果:最佳的发酵培养条件组合为温度37℃、葡萄糖20克/升、大豆胨15克/升、pH值7.3,最佳的纯化条件组合为冷酚抽提三次、沉核酸乙醇浓度23%、超滤膜孔径50kD、最终沉糖乙醇浓度60%,在此筛选得到的最佳条件下,连续进行了5个批次肺炎链球菌5型的发酵与荚膜多糖提取,荚膜多糖粗糖的平均收率为808.6mg/L,相对标准偏差为3.84%。结论:上述发酵培养条件组合适合用于肺炎多糖疫苗的研究和生产。     

Sedimentation and viscosity studies on the capsular and somatic polysaccharides of pneumococcus Type III

Sedimentation constants at infinite dilution have been found to be 1.89 and 4.06 for the somatic and capsular polysaccharides, respectively, from pneumococcus Type III. Intrinsic viscosities have been determined for the somatic and capsular polysaccharides of pneumococcus Type III using the Ostwald viscometer. Molecular weights and dimensions have been calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be prolate ellipsoids of revolution. Values for the somatic polysaccharide are: molecular weight, 26,400; diameter, 0.97 mmicro; and length, 36.18 mmicro. Values for the capsular polysaccharide are: molecular weight, 171,800; diameter, 1.04 mmicro; and length, 177.87 mmicro. The molecular weights were calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be flexible chains. The value of the molecular weight of the somatic polysaccharide is 31,500 and the value for the molecular weight of the capsular polysaccharide is 267,500. The molecules of both the somatic and capsular polysaccharides exhibit high degrees of asymmetry.     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: I. Production of the Adjuvant Polysaccharide by Noncapsulated Mutant

In culture fluid, Klebsiella pneumoniae type 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type-specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type-specific capsular polysaccharide was isolated from ultraviolet-irradiated cells of K. pneumoniae type 1 Kasuya strain which had been labeled with leucine-requiring marker by selecting unagglutinable cells with the antiserum to the type-specific capsular polysaccharide. Serological tests showed that the type-specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniae was discussed.     

细菌荚膜多糖

随着分子生物学、糖化学和免疫学的发展,细菌荚膜多糖的研究逐步深入.不仅对其特性及组成有了进一步的了解,而且对其多糖合成相关基因、合成调节和致病性进行了更为细致的研究.本文概括了细菌荚膜多糖的化学结构、合成相关基因、多样性产生机制、合成调节、功能与致病性和应用前景,总结其研究热点,以期为荚膜多糖的研究和应用提供理论依据和思路.     

The role of the capsular polysaccharide in the activation of the alternative pathway by the pneumococcus.

Previous studies have shown that when pneumococci are incubated in normal, nonimmune serum, they activate the alternative pathway and opsonically active C3b is fixed to the surface of the organism. Other studies have demonstrated that C3-dependent opsonization via the alternative pathway plays a significant role in the nonimmune host's defense against the pneumococcus. The present studies concern the role of the capsular polysaccharide in initiating the activation of the alternative pathway by the pneumococcus. Some pneumococcal capsular polysaccharide types, but not all, are able to activate the alternative pathway. Soluble purified capsular polysaccharide types 1, 4 and 25 activate the alternative pathway, whereas types 2, 3, 14, and 19 do not. Since the capsular polysaccharides exist in their native form attached to the pneumococcal surface, selected capsular polysaccharides were also tested for their ability to activate the alternative pathway when attached to a particulate carrier, sheep erythrocytes. Capsular polysaccharide types 2 and 3 failed to activate the alternative pathway when attached to sheep erythrocytes, paralleling the results obtained when these capsular polysaccharides were in solution. In contrast, the type 25 capsular polysaccharide not only activated the alternative pathway when attached to sheep erythrocytes, as it had when in solution, but it also initiated alternative pathway-mediated lysis of the erythrocytes. The capsular polysaccharide is not required for the activation of the alternative pathway by the pneumococcus. Although all types of encapsulated pneumococci are able to activate the alternative pathway, not all the purified capsular polysaccharide types are able to do so. In addition, a nonencapsulated pneumococcus, derived originally from a type 2 organism, activates the alternative pathway as well as a fully encapsulated type 2 pneumococcus.     

Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.     

Staphylococcus aureus growth and type 5 capsular polysaccharide production in synthetic media

The production of type 5 capsular polysaccharide by Staphylococcus aureus in synthetic media was investigated. The influence of medium components on capsular polysaccharide synthesis appeared to relate to the presence or absence of the component rather than to concentration gradient. The production of type 5 capsular polysaccharide was linked to energy availability and energy source, but not to carbohydrate concentration or carbon/nitrogen ratio. Regulation of capsular polysaccharide production by S. aureus in response to medium changes would appear to differ from that typically displayed in other organisms that produce polysaccharides.     

Staphylococcus aureus growth and type 5 capsular polysaccharide production in synthetic media.

The production of type 5 capsular polysaccharide by Staphylococcus aureus in synthetic media was investigated. The influence of medium components on capsular polysaccharide synthesis appeared to relate to the presence or absence of the component rather than to concentration gradient. The production of type 5 capsular polysaccharide was linked to energy availability and energy source, but not to carbohydrate concentration or carbon/nitrogen ratio. Regulation of capsular polysaccharide production by S. aureus in response to medium changes would appear to differ from that typically displayed in other organisms that produce polysaccharides.     

SEDIMENTATION AND VISCOSITY STUDIES ON THE CAPSULAR AND SOMATIC POLYSACCHARIDES OF PNEUMOCOCCUS TYPE III

Sedimentation constants at infinite dilution have been found to be 1.89 and 4.06 for the somatic and capsular polysaccharides, respectively, from pneumococcus Type III. Intrinsic viscosities have been determined for the somatic and capsular polysaccharides of pneumococcus Type III using the Ostwald viscometer. Molecular weights and dimensions have been calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be prolate ellipsoids of revolution. Values for the somatic polysaccharide are: molecular weight, 26,400; diameter, 0.97 mµ; and length, 36.18 mµ. Values for the capsular polysaccharide are: molecular weight, 171,800; diameter, 1.04 mµ; and length, 177.87 mµ. The molecular weights were calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be flexible chains. The value of the molecular weight of the somatic polysaccharide is 31,500 and the value for the molecular weight of the capsular polysaccharide is 267,500. The molecules of both the somatic and capsular polysaccharides exhibit high degrees of asymmetry.