Understanding the Chemistry and Biology of Glycosylation with Glycan Synthesis.

Glycoscience research has been significantly impeded by the complex compositions of the glycans present in biological molecules and the lack of convenient tools suitable for studying the glycosylation process and its function. Polysaccharides and glycoconjugates are not encoded directly by genes; instead, their biosynthesis relies on the differential expression of carbohydrate enzymes, resulting in heterogeneous mixtures of glycoforms, each with a distinct physiological activity. Access to well-defined structures is required for functional study, and this has been provided by chemical and enzymatic synthesis and by the engineering of glycosylation pathways. This review covers general methods for preparing glycans commonly found in mammalian systems and applying them to the synthesis of therapeutically significant glycoconjugates (glycosaminoglycans, glycoproteins, glycolipids, glycosylphosphatidylinositol-anchored proteins) and the development of carbohydrate-based vaccines.

[Colonization and transmission of Haemophilus influenzae type b (Hib)].

Haemophilus influenzae type b (Hib) remains the leading cause of invasive bacterial infection in Japanese children. More than 110 countries that have included Hib conjugate vaccines in their routine vaccination programs have seen dramatical decrease in the incidence of Hib infections. In Japan, the vaccine has been introduced for voluntary immunization since December 2008 and has been provided free of charge only since January 2011. This review reports the prevalence of Hib and its clones among healthy children and pediatric patients diagnosed with invasive or non-invasive Hib infections in Sado Island, Japan. Of 25 Hib isolates collected in this surveillance, 4 genotypic patterns (ST54-gBLPACR-III, ST54-gBLNAR-I/II, ST190-gBLNAS, and ST95-gBLPACR-I/II) were detected. These STs were double or triple-locus variants of each other. Under the same antimicrobial selective pressure, high prevalence of gBLPACR strain (76.0%) was confirmed in Hib isolates, while gBLPACR prevalence in nontypeable H. influenzae was very low (5.2%). These data suggested that each ST strain may be brought into Sado Island by different routes. We note that surveillance of healthy subjects to identify Hib carriers is important to understand their role in transmission of Hib.

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium.

Haemophilus influenzae type b (Hib), a Gram-negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl-ribitol-phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake-flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (∼500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α-ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508-1519, 2017.

Haemophilus influenzae type B meningitis among children in Hanoi, Vietnam: epidemiologic patterns and estimates of H. Influenzae type B disease burden.

From March 2000 to February 2002, a population-based study of Haemophilus influenzae type b (Hib) meningitis was conducted among children less than five years of age in Hanoi, Vietnam. Children with suspected bacterial meningitis were referred to hospitals and each patient underwent standardized clinical examination and microbiologic testing. In Hanoi, 580 children were evaluated for bacterial meningitis and 23 (4%) had confirmed or probable Hib meningitis. The incidence of all Hib meningitis was 12/100,000 child-years less than five years of age and 26/100,000 child-years less than two years of age. Nationally, an estimated 1,005 children less than five years of age are hospitalized for Hib meningitis and 5,107 are hospitalized for Hib pneumonia. Among children with Hib meningitis, at least 100 will develop severe neurologic sequelae and 40 will die. These data suggest there is a substantial burden of Hib disease in Vietnam. National leaders will be provided with these data to facilitate development of national vaccination policies for children in Vietnam.

Differential utilization by Haemophilus influenzae of haemoglobin complexed to the three human haptoglobin phenotypes.

Haemophilus influenzae has an absolute requirement for heme, which may be supplied as the haemoglobin-haptoglobin complex. Utilization of haemoglobin-haptoglobin by H. influenzae is mediated by a family of proteins termed the haemoglobin-haptoglobin binding proteins (Hgps), of which a given strain may contain up to four genes. Human haptoglobin occurs in three phenotypes (1-1, 2-1 and 2-2). Using mutant derivatives of an H. influenzae type b strain that expressed single Hgps we analysed the ability of each Hgp to utilize haemoglobin complexed to the various haptoglobin phenotypes. A strain expressing only HgpB was able to utilize haemoglobin bound to all haptoglobin phenotypes significantly better than strains expressing either HgpA or HgpC.

[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.

[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.

Characterization of polysaccharide production of haemophilus influenzae Type b and its relationship to bacterial cell growth.

Haemophilus influenzae type b (Hib) causes invasive infections in infants and young children. Vaccines consisting of Hib capsular polysaccharide (polymer of ribosylribitol phosphate [PRP]) conjugated to a protein are effective in the prevention of such infections. The production of capsular polysaccharide type b was studied in three cultivation conditions: single, glucose pulse, and repeated batch. Specific polysaccharide production (Yp/x) was calculated for all experiments, showing the following values: 67 (single-batch cultivation), 71 (glucose pulse), 75 (repeated-batch cultivation, first batch), and 87 mg of PRP/g of dry cell weight (DCW) (repeated-batch cultivation, second batch). Biomass concentration reached approximately 1.8 g of DCW/L, while polysaccharide concentration was about approximately 132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Kono's analysis and Luedeking-Piret's model.

Morphological and biochemical variations of Haemophilus influenzae type b induced by pH and temperature changes.

Haemophilus influenzae type b ATCC 10211 was cultured at different temperatures (25 degrees C-49 degrees C) and pH values (5.7-8.7) either in liquid or semisolid medium. Morphological variations of individual cells were noted by optical microscopy depending upon the conditions of growth. At higher temperatures filaments were produced whereby the length of individual cells increased compared to cultures grown at 37 degrees C. Filaments were also observed at lower pH values. Culture conditions also affected colonial morphology. At low pH values colonies had an enhanced lobulated contour and were more wrinkly and rougher than at higher pH. The changes in cellular and colonial morphology were correlated with distinct outer membrane protein profiles. The changes in temperature and pH did not affect identification of the microorganism by the API system.

H. influenzae type b (Hib) vaccine--controversies.

Hib vaccine is the 8th vaccine knocking at the door to be included in the EPI the world over. However there are some controversies that need to be addressed, especially when it comes to use of this vaccine in India. It is difficult to culture Hib unless one uses sheep blood enriched media for culture. There is a lack of good community based data on Hib burden in India. This makes many feel that Hib is rare in India. However this is not true. There are many studies that have looked at this closely. Hib is a common cause of meningitis and pneumonitis in children less than 5 years old in India. There is wide spread problem of multi-drug resistance by Hib in India. Mortality of meningitis is as high as 100% if third generation cephalosporins are not used in time. Of the survivors of meningitis, 60% develop long-term sequelae. Hib vaccine is very effective and can lead to 99% reduction with mass vaccination in just 2-3 years. It is also a very safe vaccine. Of the conjugated vaccines available in India all are equally effective and safe and there is nothing to choose one over the other. There is a need to give a booster dose at 15-18 months of age. Even UK, which never gave the booster dose, is seriously thinking of changing their practice and give a booster dose. Lastly the combination vaccines of Hib with IPV, DPwT/DPaT, and Hepatitis B are safe and effective and should be encouraged to improve the compliance. The use of Hib vaccine is recommended in India, for those who can afford the vaccine.

[Cultivation of Haemophilus influenzae, serotype B, in amino peptide-based semisynthetic nutrient medium]. / Kul'tivirovanie Haemophilus influenzae serotipa B na polusinteticheskoi pitatel'noi srede na osnove aminopeptida.

The work shows the possibility of the cultivation of H. influenzae, serotype b, in semisynthetic nutrient medium with amino peptide as the only source of amino acids, glucose--as the main source of carbon and energy and containing, in addition, the necessary growth factors and vitamins.

[Dynamics of growth of Haemophilus influenzae serotype B and synthesis of capsular polysaccharide in the process of cultivation in a synthetic nutrient medium]. / Dinamika rosta Haemophilus influenzae serotipa B i sinteza kapsul'nogo polisakharida v protsesse kul'tivirovaniia na sinteticheskoi pitatel'noi srede.

The dynamics of H. influenzae, serotype b, growth and synthesis of their capsular polysaccharide in the synthetic nutrient medium, proposed by Herriot for noncapsular strains, was studied using 6 strains. The growth rate of H. influenzae, serotype b, and the amount of capsular polysaccharide, synthesized in the above mentioned medium, practically were not different from those in heart-brain broth (Difco). The possibility of minimizing the composition of Herriot's medium without any adverse effect on the amount of synthesized capsular polysaccharide was shown. As the result of these studies, the expediency of the cultivation of H. influenzae, serotype b, in the synthetic medium, intended for obtaining the preparations of capsular polysaccharide, was proved.

[Specific characteristics of composition of synthetic nutrient media for cultivation of Hemophilus influenzae type b from the standpoint of bacterial metabolism]. / Osobennosti sostava sinteticheskikh pitatel'nykh sred dlia kul'tivirovaniia Hemophilus influenzae type b s pozitsii sovremennykh predstavlenii o metabolizme étikh bakterii.

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.

Improvement in the purification process of the capsular polysaccharide from Haemophilus influenzae type b by using tangential ultrafiltration and diafiltration.

Capsular polysaccharide produced by Haemophilus influenzae b (Hib) is the main virulent agent and used as the antigen in the vaccine formulation. In this study, an improved process of polysaccharide purification was established based on tangential flow ultrafiltration using detergents (cocamidopropyl betaine and sodium deoxycholate), two selective ethanol precipitations steps, and extensive enzymatic hydrolysis as strategy. The relative purity (RP) related to protein and nucleic acids were 122~263 and 294~480, respectively, and compatible with the specifications established by the World Health Organization for Hib vaccine, RP≥100. These results make this process simple, cheaper, efficient, environmentally friendly, and prone to be scaled up.

Structural elucidation of lipopolysaccharide core oligosaccharides from lic1 and lic1/lic2 mutants of Haemophilus influenzae type b strain Eagan.

The structures of lipopolysaccharides (LPSs) of lic1 and lic1/lic2 mutants from Haemophilus influenzae type b strain Eagan (RM153) were investigated using methylation analysis, electrospray ionization - mass spectrometry, and nuclear magnetic resonance spectroscopy on O-deacylated, O- and N-deacylated core oligosaccharide (OS); and deacylated, dephosphorylated, and terminally reduced samples. The backbone OS derived from the major LPS glycoforms were determined to consist of the inner-core triheptosyl unit, L-alpha-D-Hepp-(1-2)-L-alpha-D-Hepp-(1-3)-L-alpha-D-Hepp-(1-, common to all H. influenzae strains investigated to date that is linked to the lipid A region of the molecule via a Kdo residue to which beta-D-Glcp and beta-D-Galp residues are attached in 1,4 and 1,2 linkages to the proximal (HepI) and distal (HepIII) heptose residues, respectively. It was found that the lic1 mutant predominately elaborates the Hex4 LPS glycoforms previously identified in the parent strain where a beta-D-Glcp-(1-4)-alpha-D-Glcp unit is linked in a 1,3 linkage to the central heptose (HepII) of the triheptosyl moiety. The lic1 locus consists of 4 genes (lic1A to lic1D) in a single transcriptional unit that directs phase variable expression of phosphocholine. The lic1A gene is phased off in the RM153 isolate of strain Eagan. LPS from the double mutant, lic1/lic2 had a similar structure to that of lic1 mutant except that there was no chain extension from the central heptose in the inner core (HepII). The lic2 locus consists of 4 genes (lic2A to lic2D). Our structural data were consistent with the proposed function of lic2C, providing the first definitive evidence for its role as the glycosyltransferase required for chain initiation from HepII. The presence of an O-acetyl group at O-3 of the distal heptose (HepIII) was elucidated by 1H NMR on the mild acid liberated core OS samples.

Within-host evolution for the invasiveness of commensal bacteria: an experimental study of bacteremias resulting from Haemophilus influenzae nasal carriage.

BACKGROUND: Many bacteria responsible for clinically relevant disease reside harmlessly in a large fraction of humans. Three explanations have been proposed to account for why these normally commensal bacteria occasionally cause invasive disease: host susceptibility, stochasticity in the host-bacteria interaction, and the evolution of invasive mutants in colonized hosts. Here we test the third of these hypotheses for the rare invasiveness of commensal bacteria: within-host evolution. METHODS AND RESULTS: Using neonatal rats intranasally colonized with pairs of marked Haemophilus influenzae type b strains, we demonstrate that the resulting bacteremias are derived from single organisms. To test the within-host evolution hypothesis we explored the relative ability of bacteria isolated from the blood and nasal passages of bacteremic rats to colonize the nasopharynx and invade the bloodstream. CONCLUSIONS: Our results provide support for within-host evolution as one but not the sole explanation for the invasiveness of these bacteria. We discuss the implications of these results for both the rare invasiveness of commensal bacteria and the general observation that bacteria isolated from the sites of human invasive disease are almost invariably monoclonal.

Relationships among physico-chemical and biological tests for a synthetic Hib-TT conjugate vaccine.

The relationships among physico-chemical and biological tests were evaluated in this study for Cuban Hib vaccine, a tetanus toxoid conjugated with a polysaccharide obtained from chemical synthesis, in order to estimate the biological significance of the relevant physico-chemical assays for lot released. In order to get samples with theoretical different potencies, Hib-TT samples were stored at -20, 4, 25, 37 and 70 degrees C for 5 weeks and after they were evaluated by HPLC and Orcinol methods and for immunogenicity and bactericidal activity. We found strong relationships between HPLC and free PRP level (r2=0.9571), anti-PRP antibodies and bactericidal activity (r2=0.9649) and free PRP content and anti-PRP antibodies titres (r2=0.7671). A lower correlation was found for HPLC and anti-PRP titres (r2=0.5996). We demonstrate that it is possible to monitor this new product combining physico-chemical and biological tests in order to contribute to its characterisation.

Haemophilus influenzae surface fibrils contribute to serum resistance by interacting with vitronectin.

Vitronectin inhibits the membrane attack complex of the complement system and is found both in plasma and the extracellular matrix. In this study, we have identified the outer membrane protein Haemophilus surface fibrils (Hsf) as the major vitronectin-binding protein in encapsulated H. influenzae type b. A H. influenzae mutant devoid of Hsf showed a significantly decreased binding to both soluble and immobilized vitronectin as compared with the wild-type counterpart. Moreover, Escherichia coli-expressing Hsf at the surface strongly adhered to immobilized vitronectin. Importantly, the H. influenzae Hsf mutant had a markedly reduced survival as compared with the wild-type bacterium when incubated with normal human serum. A series of truncated Hsf fragments were recombinantly manufactured in E. coli. The vitronectin binding regions were located within two separate binding domains. In conclusion, Hsf interacts with vitronectin and thereby inhibits the complement-mediated bactericidal activity, and thus is a major H. influenzae virulence factor.

Nasopharyngeal colonization with Haemophilus influenzae type b among infants and children in Japan.

Healthy carriers of Haemophilus influenzae type b (Hib) play an important role in the spread of invasive Hib disease. The aim of the present study was to estimate Hib colonization among infants and children in Japan. Specimens from throat and nasopharyngeal cultures were obtained by thorough swabbing of both tonsils and the posterior pharynx. Specimens were inoculated on Hib antiserum agar. This was prepared with Levinthal base and Hib antiserum. Conventional methods were used concomitantly. Four of 474 infants from 1-48 months of age (0.84%) had Hib cultured from their nasopharynx. The carriage rate in 1-12 months old infants was 0.62% (2/322 cases), and that in 13-48 month old children was 1.32% (2/152 cases). Five of 167 (3.0%) 13-year-old children, and five of 154 (3.2%) 9-year-old children were asymptomatic carriers. Thirty-five of 104 household contacts of a patient with invasive Hib disease (33.6%) had Hib colonization. The carriage rate in healthy Japanese children may not be different from that in the USA prior to the availability of the conjugate Hib vaccine. The Hib carriage rate in household contacts of patients with invasive Hib disease was higher than in healthy children (P < 0.005). Our results suggest the possibility of an outbreak of invasive Hib disease in Japan.

Interaction of fimbriae of Haemophilus influenzae type B with heparin-binding extracellular matrix proteins.

The interaction of the fimbriae of Haemophilus influenzae type b (Hib) with two heparin-binding extracellular matrix proteins, human fibronectin (Fn) and heparin-binding growth-associated molecule (HB-GAM) from mouse, were studied. The fimbriated Hib strain 770235 fim+, as well as the recombinant strain E. coli HB101(pMH140), which expressed Hib fimbriae, adhered strongly to Fn and HB-GAM immobilized on glass. Purified Hib fimbriae bound to Fn and HB-GAM, and within the Fn molecule, the binding was localized to the N-terminal 30,000-molecular-weight (30K) and 40K fragments, which contain heparin-binding domains I and II, respectively. Fimbrial binding to Fn, HB-GAM, and the 30K and the 40K fragments was inhibited by high concentrations of heparin. The results show that fimbriae of Hib interact with heparin-binding extracellular matrix proteins. The nonfimbriated Hib strain 770235 fim- exhibited a low level of adherence to Fn but did not react with HB-GAM, indicating that Hib strains also possess a fimbria-independent mechanism to interact with Fn.