Free Saccharide Research Articles

Analytical Challenges in Novel Pentavalent Meningococcal Conjugate Vaccine (A, C, Y, W, X)

Multivalent meningococcal conjugate vaccines are a significant focus for the scientific community in light of the WHO’s mission to defeat meningitidis by 2030. Well-known meningococcal vaccines such as MenAfriVac, Nimenrix, Menveo, and MenQuadfi are licensed in various parts of the world and have been successful. Recently, the World Health Organization (WHO) qualified MenFive (meningococcal A, C, Y, W, and X) conjugate vaccine, further enhancing the battery of vaccines against meningitis. The antigenic nature of the current and new serogroups, the selection of carrier proteins, and the optimal formulation of these biomolecules are pivotal parameters for determining whether a biological preparation qualifies as a vaccine candidate. Creating appropriate quality control analytical tools for a complex biological formulation is challenging. A scoping review aims to identify the main challenges and gaps in analyzing multivalent vaccines, especially in the case of novel serogroups, such as X, as the limited literature addresses these analytical challenges. In summary, the similarities in polysaccharide backbones between meningococcal serogroups (C, Y, W sharing a sialic backbone and A, X sharing a phosphorous backbone) along with various conjugation chemistries (such as CNBr activation, reductive amination, CDAP, CPIP, thioether bond formation, N-hydroxy succinimide activation, and carbodiimide-mediated coupling) resulting into a wide variety of polysaccharide -protein conjugates. The challenge in analyzing carrier proteins used in conjugation (such as diphtheria toxoid, tetanus toxoid, CRM diphtheria protein, and recombinant CRM) is assessing their purity (whether they are monomeric or polymeric in nature as well as their polydispersity). Additional analytical challenges include the impact of excipients, potential interference from serogroups, selection and establishment of standards, age-dependent behavior of biomolecules indicated by molecular size distributions, and process-driven variations. This article explains the analytical insights gained (polysaccharide content, free saccharide, free proteins, MSD) during the development of the MenFive vaccine and highlights the crucial gaps and challenges in testing.

A New Experimental Setup to Characterize Binder–Vegetal Particle Compatibility in Plant-Based Concrete

The good insulation properties and the low carbon footprint of vegetal concretes make them promising materials whose use tends to grow continuously. To produce optimized building materials, a better understanding of the interfacial transition zone (ITZ) between vegetal particles and cement paste in terms of the reactions involved and the size of the impacted surface was investigated. This research led to the setting of a reliable visual test to observe ITZ, which enables the monitoring of its appearance and development. Different combinations of vegetal particles and cement pastes were tested to compare the formed ITZ: hemp, rapeseed, and bamboo into Portland and Prompt cement. Finally, a clear link was drawn between the sugar concentration and the size of ITZ. Thanks to image analysis, it was shown that ITZ is due to physico-chemical reactions, with the extraction of free saccharide molecules from the vegetal and water suction followed by their release into the cement paste.

Multi-Glycomic Characterization of Fiber from AOAC Methods Defines the Carbohydrate Structures.

Dietary fiber has long been known to be an essential component of a healthy diet, and recent investigations into the gut microbiome-health paradigm have identified fiber as a prime determinant in this interaction. Further, fiber is now known to impact the gut microbiome in a structure-specific manner, conferring differential bioactivities to these specific structures. However, current analytical methods for food carbohydrate analysis do not capture this important structural information. To address this need, we utilized rapid-throughput LC-MS methods to develop a novel analytical pipeline to determine the structural composition of soluble and insoluble fiber fractions from two AOAC methods (991.43 and 2017.16) at the total monosaccharide, glycosidic linkage, and free saccharide level. Two foods were chosen for this proof-of-concept study: oats and potato starch. For oats, both AOAC methods gave similar results. Insoluble fiber was found to be comprised of linkages corresponding to β-glucan, arabinoxylan, xyloglucan, and mannan, while soluble fiber was found to be mostly β-glucan, with small amounts of arabinogalactan. For raw potato starch, each AOAC method gave markedly different results in the soluble fiber fractions. These observed differences are attributable to the resistant starch content of potato starch and the different starch digestion conditions used in each method. Together, these tools are a means to obtain the complex structures present within dietary fiber while retaining "classical" determinations such as soluble and insoluble fiber. These efforts will provide an analytical framework to connect gravimetric fiber determinations with their constituent structures to better inform gut microbiome and clinical nutrition studies.

Development of a New Solid-Phase Extraction Base Method for Free Saccharide Content Estimation of Meningococcal Conjugate Vaccines.

GlaxoSmithKline (GSK) is currently developing a fully liquid presentation to ease the administration of the licensed quadrivalent conjugate vaccine (Menveo) against meningococcal serogroup A, C, W, and Y (MenACWY) infections. Herein, we report a new method for determining the free saccharide (FS) content of CRM197-MenACWY conjugated antigens, with the aim of improving accuracy and reproducibility. Mathematical models have been used to support technical knowledge in reducing the need for experimental development. This results in an improved, faster, and platform-based technique for FS separation with one single pretreatment applicable to all antigens of the multivalent meningococcal vaccine.

Clinically proven specification setting for a meningococcal serogroup a conjugate vaccine

GSK is currently working to improve the commercial presentation of the licensed quadrivalent conjugate vaccine (Menveo) for use against meningococcal serogroup A, C, W, Y (MenACWY) infections. Menveo consists of a primary, lyophilized vial, containing the serogroup A antigen that is reconstituted with the content of a second, liquid, vial that contains the serogroup C, W, Y antigens, to give the final liquid MenACWY product.Since the MenA structure is prone to hydrolytic degradation in liquid formulations, we used mathematical models to rationally design a clinical Phase 2 development plan and provide end of shelf-life (EoSL) and release specification setting for the MenACWY liquid product.By using development and clinical stability data, statistical models were built and used to predict both the MenA free saccharide (FS) and O-Acetyl (OAc) content during long-term storage conditions at 5 °C and stressed (accelerated) stability studies at 15 °C, 22.5 °C, 25 °C, 37 °C and 50 °C. This approach allowed us to define an aging plan for the clinical material to reach at least the required levels of MenA FS and OAc levels at product EoSL. The clinical material was then exposed to a temperature of 22.5 ± 2.5 °C for 59 days to generate FS OAc content of about 35% and 40%, respectively, which was then delivered to the patients in the clinical trial.To the best of our knowledge, this work represents the first example in the field of vaccine research where statistical models have been used to rationally design tailored lots, with the goal of setting EoSL and release specification limits based on data collected on artificially aged clinical material, in which the FS and OAc levels tested were intended to support a product shelf-life of at least 24 months.

Glycoconjugate content quantification to assess vaccine potency: A simplified approach

Several glycoconjugate vaccines have been licensed or are currently in clinical development to prevent bacterial infections.Here we report the development of a single analytical assay to quantify the conjugated saccharide content, as alternative to two separated total and free (unconjugated) saccharide assays used so far, for a quadrivalent conjugate vaccine containing meningococcal serogroup A polysaccharide (α-1,6-linked N-acetylmannosamine phosphate repeating unit partly O-acetylated at position C3 or C4) coupled with CRM197 protein.The results confirm a high linear correlation among the two approaches (conjugated saccharide content vs. difference of total saccharide and free saccharide).Conjugated saccharide content estimation is therefore demonstrated to be a suitable method to monitor the product quality of vaccines containing meningococcal serogroup A conjugate antigen, in the final filled presentation as demonstrated here and potentially on the bulk conjugate before formulation.

632. Clinical Experience with a New Fully Liquid Presentation of the MenACWY-CRM Vaccine. Results from Two Multicenter, Randomized, Controlled, Observer-Blind, Phase 2b Studies

Abstract Background Currently, licensed MenACWY-CRM conjugate vaccine presentation (Lyo/Liq) consists of two vials (lyophilized MenA, liquid MenCWY) to be reconstituted before injection. A new, fully liquid, single vial formulation has been developed and evaluated in two clinical studies in adolescents and adults aimed at demonstrating immunological non-inferiority of the liquid presentation for MenA. Methods Overall, 1337 subjects, 10 to 40 years of age (y), were exposed to a single 0.5 mL intramuscular dose of MenACWY Liquid and 1332 to MenACWY-CRM(Lyo/Liq). MenACWY-CRM Liquid was aged before administration, to test the vaccine immunogenicity at the end of the intended shelf-life and establish release and end of shelf life specifications. MenACWY-CRM(Lyo/Liq) was used as comparator and was not aged. In study 1 (NCT03652610), the Liquid vaccine underwent an ageing process under controlled conditions to reach ~30% MenA free saccharide (FS). In study 2 (NCT03433482), the Liquid vaccine was naturally aged at 2–8°C for approximately 24 and 30 months. Primary immunogenicity objective in both studies was non-inferiority of MenACWY-CRM liquid to licensed vaccine, as measured by human serum bactericidal assay (hSBA) geometric mean titers (GMTs) against MenA, 1-month post-vaccination. Results In both studies, for each between-group ratio of MenA hSBA GMTs, lower limits of the 95% confidence intervals (CIs) were greater than the prespecified non-inferiority margin (0.5), thus meeting the non-inferiority immunogenicity objective. Irrespectively of the vaccine presentation tested, over 82% of participants achieved MenA hSBA titers ≥ 8 in study 1 and at least 92% in study 2. The immunogenicity of MenACWY-CRM Liquid was similar to that of MenACWY-CRM(Lyo/Liq) when analyzed by serogroup, overall. No related serious adverse events were reported for both presentations. Conclusion After ageing, the new MenACWY-CRM Liquid demonstrated the ability to elicit non-inferior bactericidal responses against MenA compared to licensed formulation. The new full-liquid presentation is expected to increase the user-friendliness of the vaccine as well as to reduce reconstitution errors in the future, with a similar safety profile to that of the licensed vaccine presentation. Disclosures Javier Díez-Domingo, MD, PhD, GSK (Grant/Research Support)Sanofi (Grant/Research Support) Corinne Vandermeulen, MD PhD, GSK (Grant/Research Support)MSD (Grant/Research Support)Pfizer (Grant/Research Support) Tauseefullah Akhund, MD, MSc, MPH, GSK (Employee, Shareholder) Marco Costantini, MSc, MBA, EMPHA, GSK (Employee) Puneet Vir Singh, MBBS, PGDCD, GSK (Employee, Shareholder) Venere Basile, MSc, GSK (Employee, Shareholder) Elena Fragapane, MD, GSK (Employee, Shareholder) Maria Lattanzi, MD, PhD, GSK (Employee, Shareholder) Michele Pellegrini, MD, PhD, Michele Pellegrini (Employee, Shareholder)

Immunological non-inferiority of a new fully liquid presentation of the MenACWY-CRM vaccine to the licensed vaccine: results from a randomized, controlled, observer-blind study in adolescents and young adults

ABSTRACT A fully liquid MenACWY-CRM vaccine presentation has been developed, modifying the meningococcal serogroup A (MenA) component from lyophilized to liquid. The safety and immunogenicity of the liquid presentation at the end of the intended shelf-life (aged for 24 or 30 months) were compared to the licensed lyophilized/liquid presentation. This multicenter, randomized (1:1), observer-blind, phase 2b study (NCT03433482) enrolled adolescents and young adults (age 10–40 years). In part 1, 844 participants received one dose of liquid presentation stored for approximately 24 months or licensed presentation. In part 2, 846 participants received one dose of liquid presentation stored for approximately 30 months or licensed presentation. After storage, the MenA free saccharide (FS) level was approximately 25% and O-acetylation was approximately 45%. The primary objective was to demonstrate non-inferiority of the liquid presentation to licensed presentation, as measured by human serum bactericidal assay (hSBA) geometric mean titers (GMTs) against MenA, 1-month post-vaccination. Immune responses against each vaccine serogroup were similar between groups. Between-group ratios of hSBA GMTs for MenA were 1.21 (part 1) and 1.11 (part 2), with two-sided 95% confidence interval lower limits (0.94 and 0.87, respectively) greater than the prespecified non-inferiority margin (0.5), thus meeting the primary study objective. No safety concerns were identified. Despite reduced O-acetylation of MenA and increased FS content, serogroup-specific immune responses induced by the fully liquid presentation were similar to those induced by the licensed MenACWY-CRM vaccine, with non-inferior anti-MenA responses. The safety profiles of the vaccine presentations were similar.

A new fully liquid presentation of MenACWY-CRM conjugate vaccine: Results from a multicentre, randomised, controlled, observer-blind study

BackgroundThe currently licensed quadrivalent MenACWY-CRM conjugate vaccine presentation consists of two vials (lyophilised MenA and liquid MenCWY) to be reconstituted before injection. A new fully liquid formulation in a single vial has been developed to further improve the vaccine presentation. Since the MenA structure is subject to hydrolytic degradation, this study was conducted to compare the immunogenicity and safety of the investigational MenACWY-CRM liquid vaccine with the licensed vaccine. MethodsIn this multicentre, randomised, controlled, observer-blind, phase 2b study, 979 healthy adults were administered a single dose of MenACWY-CRM liquid presentation or the currently licensed MenACWY-CRM vaccine. MenA free saccharide generation was accelerated to approximately 30% in the liquid presentation and MenA polysaccharide O-acetylation was reduced to approximately 40%, according to a controlled procedure. Immunological non-inferiority of the MenACWY-CRM liquid to the licensed vaccine, as measured by human serum bactericidal assay (hSBA) geometric mean titres (GMTs) against MenA 1 month post-vaccination, was the primary study objective. Safety assessment was among the secondary objectives. ResultsImmune responses against each serogroup were similar between the two vaccine groups and was non-inferior for MenA. Adjusted hSBA GMTs for MenA were 185.16 and 211.33 for the MenACWY-CRM liquid presentation and currently licensed vaccine presentation, respectively. The between-group ratio of hSBA GMTs for MenA was 0.88, with a two-sided 95% confidence interval lower limit of 0.64, greater than the prespecified non-inferiority margin of 0.5, thus meeting the primary study objective. Both vaccines were well tolerated. No serious adverse events were considered related to vaccination. ConclusionsThe levels of MenA free saccharide and polysaccharide O-acetylation did not affect the immunogenicity of the fully liquid presentation, which was demonstrated to be non-inferior to the immunogenicity of the currently licensed MenACWY-CRM vaccine against MenA. The immunogenicity, reactogenicity and safety profiles of the two vaccine presentations were similar.

Evidence of Extended Thermo-Stability of Typhoid Polysaccharide Conjugate Vaccines.

Typhoid conjugate vaccines (TCV) are effective in preventing enteric fever caused by Salmonella enterica serovar Typhi in Southeast Asia and Africa. To facilitate vaccination with the Vi capsular polysaccharide–tetanus toxoid conjugate vaccine, Typbar TCV, and allow it to be transported and stored outside a cold chain just prior to administration, an extended controlled-temperature conditions (ECTC) study was performed to confirm the quality of the vaccine at 40 °C for 3 days at the end of its shelf-life (36 months at 2–8 °C). Studies performed in parallel by the vaccine manufacturer, Bharat Biotech International Limited, and an independent national control laboratory (NIBSC) monitored its stability-indicating parameters: O-acetylation of the Vi polysaccharide, integrity of the polysaccharide–protein conjugate, and its molecular size and pH. ECTC samples stored at 40 °C and 45 °C in comparison with control samples stored at 4 °C and 55 or 56 °C, were shown to have stable O-acetylation and pH; only very slight increases in the percentage of free saccharide and corresponding decreases in molecular size were observed. The deoxycholate method for precipitating conjugated polysaccharide was very sensitive to small incremental increases in percentage of free saccharide, in line with storage temperature and duration. This extended ECTC study demonstrated minimal structural changes to the Vi polysaccharide and conjugate vaccine and a stable formulation following extended exposure to elevated temperatures for the desired durations. This outcome supports the manufacturer’s ECTC claim for the vaccine to be allowed to be taken outside the cold chain before its administration.

Evaluation of Critical Quality Attributes of a Pentavalent (A, C, Y, W, X) Meningococcal Conjugate Vaccine for Global Use.

Towards achieving the goal of eliminating epidemic outbreaks of meningococcal disease in the African meningitis belt, a pentavalent glycoconjugate vaccine (NmCV-5) has been developed to protect against Neisseria meningitidis serogroups A, C, Y, W and X. MenA and X polysaccharides are conjugated to tetanus toxoid (TT) while MenC, Y and W polysaccharides are conjugated to recombinant cross reactive material 197 (rCRM197), a non-toxic genetic variant of diphtheria toxin. This study describes quality control testing performed by the manufacturer, Serum Institute of India Private Limited (SIIPL), and the independent control laboratory of the U.K. (NIBSC) on seven clinical lots of the vaccine to ensure its potency, purity, safety and consistency of its manufacturing. In addition to monitoring upstream-manufactured components, samples of drug substance, final drug product and stability samples were evaluated. This paper focuses on the comparison of the vaccine’s critical quality attributes and reviews key indicators of its stability and immunogenicity. Comparable results were obtained by the two laboratories demonstrating sufficient levels of polysaccharide O-acetylation, consistency in size of the bulk conjugate molecules, integrity of the conjugated saccharides in the drug substance and drug product, and acceptable endotoxin content in the final drug product. The freeze-dried vaccine in 5-dose vials was stable based on molecular sizing and free saccharide assays. Lot-to-lot manufacturing consistency was also demonstrated in preclinical studies for polysaccharide-specific IgG and complement-dependent serum bactericidal activity for each serogroup. This study demonstrates the high quality and stability of NmCV-5, which is now undergoing Phase 3 clinical trials in Africa and India.

Aqueous synthesis of sialylglycopeptide‐grafted glycopolymers with high affinity for the lectin and the influenza virus hemagglutinin

ABSTRACTGlycopolymers with pendant complex‐type sialyl N‐glycans containing heptapeptides, that is, sialylglycopeptides (SGPs), were synthesized using a water soluble polymer backbone bearing N‐hydroxysulfosuccinimidyl esters by post‐polymerization modification in water. Although SGP has three amino groups on the peptide chain, the substitution reaction occurs preferentially at the N‐terminus α‐amino group in the lysine residue onto the polymer side chain because the reactivity of such α‐amino group is higher than that of the ε‐amino group in the lysine residue under mild acidic aqueous condition. The resulting SGP‐grafted glycopolymers exhibited strong interaction with the lectin Sambucus sieboldiana agglutinin and the human influenza A virus hemagglutinin, with higher binding associate constant values than those of free saccharide according to quartz crystal microbalance analysis. © 2020 Wiley Periodicals, Inc. J. Polym. Sci. 2020, 58, 548–556

Brazilian meningococcal C conjugate vaccine: physicochemical, immunological, and thermal stability characteristics.

High temperature is known to cause some instability in polysaccharide-protein conjugated vaccines and studies under stress conditions may be useful in determining whether short-term accidental exposure to undesired conditions can compromise product quality. In this study, we examined the structural stability of three industrial batches of Brazilian Meningococcal C conjugate bulk (MPCT) incubated at 4, 37, and 55°C for 5weeks. The effect of exposure to the storage temperatures was monitored by HPLC-SEC, CZE, CD and NMR techniques. The immunological significance of any physicochemical changes observed in MPCT was determined by SBA and ELISA assays of serum from immunized mice. Fluorescence emission spectra at 4 and 37°C were similar among all samples and compatible with the native fold of the carrier protein. Fluorescence spectra of MPCT stored at 55°C decreased in intensity and had a significant red-shift, indicating conformational changes. Far-UV CD spectra revealed a trend toward loss of structural conformation as storage temperature was increased to 55°C. The NMR data showed modified signal intensity of the aromatic and aliphatic residues, mainly for samples incubated at 55°C, suggesting a partial loss of tertiary structure. About 50% free saccharide content was found in bulks stored at 55°C, but no difference was observed in the IgG or SBA titers. The present study showed physicochemical methods alone are insufficient to predict the biological activity of a MPCT conjugate vaccine without extensive validation against immunological data. However, they provide a sensitive means of detecting changes induced in a vaccine exposed to adverse environmental condition.

Development and characterization of Haemophilus influenzae type b conjugate vaccine prepared using different polysaccharide chain lengths

Capsular polysaccharide conjugates of Haemophilus influenzae type b (Hib) are important components of several mono- or multi-valent childhood vaccines. However, their access to the most needy people is limited due to their high cost. As a step towards developing a cost effective and more immunogenic Hib conjugate vaccine, we present a method for the preparation of Hib capsular polysaccharide (PRP)–tetanus toxoid (TT) conjugates using optimized PRP chain length and conjugation conditions. Reactive aldehyde groups were introduced into the polysaccharides by controlled periodate oxidation of the native polysaccharide, which were subsequently covalently linked to hydrazide derivatized tetanus toxoid by means of reductive amination. Native polysaccharides were reduced to average 100 or 50kDa polysaccharide and 10kDa oligosaccharides in a controlled manner. Various conjugates were prepared using Hib polysaccharide and oligosaccharide yielding conjugates with polysaccharide to protein ratios in the range of 0.25–0.5 (w/w) and free saccharide levels of less than 10%. Immunization of Sprague Dawley rats with the conjugates elicited specific antibodies to PRP. The low molecular weight PRP–TT conjugates were found to be more immunogenic as compared to their high molecular weight counterparts and the PRP–TT reference vaccine.

A physico-chemical assessment of the thermal stability of pneumococcal conjugate vaccine components

Physico-chemical analysis of pneumococcal polysaccharide (PS)-protein conjugate vaccine components used for two commercially licensed vaccines was performed to compare the serotype- and carrier protein-specific stabilities of these vaccines. Nineteen different monovalent pneumococcal conjugates from commercial vaccines utilizing CRM197, diphtheria toxoid (DT), Protein D (PD) or tetanus toxoid (TT) as carrier proteins were incubated at temperatures up to 56°C for up to eight weeks or were subjected to freeze-thawing (F/T). Structural stability was evaluated by monitoring their size, integrity and carrier protein conformation. The molecular size of the vaccine components was well maintained for Protein D, TT and DT conjugates at -20°C, 4°C and F/T, and for CRM197 conjugates at 4°C and F/T. It was observed that four of the eight serotypes of Protein D conjugates tended to form high molecular weight complexes at 37°C or above. The other conjugated carrier proteins also appeared to form oligomers or ‘aggregates’ at elevated temperatures, but rarely when frozen and thawed. There was evidence of degradation in some of the conjugates as evidenced by the formation of lower molecular weight materials which correlated with measured free saccharide. In conclusion, pneumococcal-Protein D/TT/DT and most CRM197 bulk conjugate vaccines were stable when stored at 2–8°C, the recommended temperature. In common between the conjugates produced by the two manufacturers, serotypes 1, 5, and 19F were relatively less stable and 6B was the most stable, with types 7F and 23F also showing good stability.

Preparation of bacterial polysaccharide–protein conjugates: Analytical and manufacturing challenges

A conjugate can be a polysaccharide (PS) covalently attached to a protein, which provides T cell epitopes for a normally T cell independent antigen. To produce a conjugate vaccine, the purified PS must first be chemically modified to generate reactive groups that can link to the protein. Two commonly used methods for PS activation are periodate oxidation at vicinal hydroxyls and cyanylation of hydroxyls. The PS should be of known molecular size before and after activation. Low molecular weight impurities in the protein may result in inefficient conjugation. Two critical measures after conjugation and purification are the PS to protein ratio and the percent non-conjugated saccharide (free saccharide). Yield and conjugate stability are critical considerations. Typically, considerably less than 20% of the activated PS becomes conjugated. Yield can be improved using newer conjugation methods, whereby highly reactive groups are generated on both the PS and carrier protein with yields approaching 50%. Two major measures used to follow vaccine stability are changes in molecular size and percent free (unbound) PS.

A Retrospective Study on the Quality of Haemophilus Influenzae Type B Vaccines Used in the U.K. Between 1996 and 2004

Following the reduction in efficacy of Hib-TT vaccines in the primary immunization schedule observed in the U.K. between 1999 and 2003, batches of vaccine manufactured by two different companies were retrospectively examined by the National Institute for Biological Standards and Control. The study evaluated 41 batches of the Hib-TT vaccines manufactured between 1994 and 2003, assaying potency (total PRP saccharide content), integrity (% free saccharide), consistency (molecular sizing), and immunogenicity, as well as reviewing data previously obtained at the time of release. The study indicated the stability of the lyophilized final fill vaccines to extend well past their assigned shelf-lives, and found no trends in the endotoxin content, total saccharide or % free saccharide content. A trend towards slightly larger conjugates was observed over time in Hib-TT A, evidenced in both the manufacturer’s data obtained at the time that samples were submitted for testing and in data obtained from the retrospective analysis. The study confirmed that that there had been no significant change in the quality of the Hib vaccines that could possibly account for the change reported in their protective efficacy in the U.K. The study also demonstrated the value of independent testing of vaccines from the time of licensure and in the ongoing monitoring and re-examination of selected batches, as necessary, to assure their continuing quality, safety and consistency.

Glycohistochemistry of a marine sponge, Chondrilla nucula (Porifera, Desmospongiae), with remarks on a possibly related antimicrobial defense strategy and a note on exopinacoderm function

Based on carbohydrate histochemistry, including the use of lectins, and TEM, the study describes the distribution of terminal sugars in different structures of the demosponge Chondrilla nucula. The results of the general and specific carbohydrate histochemical approaches confirmed the presence of acidic and neutral glycoconjugates in the cells, and, with declining amounts from the ectosome to the mesohyl, in the extracellular matrix (ECM). AB-PAS staining indicated acidic complex carbohydrates particularly in the exopinacoderm, and more neutral ones in the cells and the ECM of the mesohyl. The PO-lectins applied demonstrated a general spectrum of free saccharide residues (α-d-mannose, α-/β-d-N-acetylglucosamine, α-d-N-acetylgalactosamine, α-d-galactose, β-d-galactose) in both sponge parts; α-l-fucose was only distinct in the ectosome. Sialic acids [siaα(2,3)-galactose, siaα(2,6)-N-acetylgalactosamine] were dominant in the very thin exopinacoderm, indicating O-linked high molecular weight glycoproteins. In this way a glycophysiologically ‘rigid’ outer mucus cover is developed as protection against mechanical hazards. Some of the free sugars (α-d-mannose, N-acetylglucosamine, N-acetylgalactosamine β-d-galactose, α-l-fucose) are known to prevent the adherence of different bacteria and fungi to cellular surfaces. Thus a high concentration of such sugars, may impede massive attacks of micro-inhabitants on mobile sponge cells, pinacocytes, and the exopinacoderm layer.

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 expresses the monofucosyl A type 1 histo-blood group epitope.

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 was obtained by phenol-water extraction of bacterial cells. Structural investigations were made on the lipid A free saccharide moiety released from the lipopolysaccharide by mild acetic acid hydrolysis. Nuclear magnetic resonance, gas liquid chromatography-mass spectrometry and fast atom bombardment-mass spectrometry were employed in the characterization of products from chemical manipulations. A monoclonal antibody specific for blood group A reacted strongly with lipopolysaccharide of H. mustelae. Chemical and serological data showed that the outer core region of the lipopolysaccharide from H. mustelae ATCC 43772 expresses the monofucosyl A type 1, alpha-D-GalNAc-(1-->3)-[alpha-L-Fuc-(1-->2]-beta-D-Gal-(1-->3)- beta-D-GlcNAc, blood group determinant, a mimic of animal cell surface glycolipids and glycoproteins.

An integrity assay for a meningococcal type B conjugate vaccine

The development of an analytical procedure for the evaluation of a conjugate vaccine’s structural wholeness or integrity is described. The principle component of the vaccine was the N-propionylated group B meningococcal polysaccharide (NPr-GBMP) covalently attached to a carrier protein. The goal of the procedure was to determine whether any whole polysaccharide, oligosaccharide, or monosaccharide, from minute to moderate levels, became detached off the conjugate. Free saccharide was isolated from the formulation, which included an aluminum hydroxide adjuvant for analysis. Due to its linkage, the NPr-GBMP did not release sialic acid efficiently with acid hydrolysis to the extent necessary for accurate quantitation. To accomplish depolymerization, the NPr-GBMP was subjected to methanolysis, 3 N hydrochloric acid in methanol for 16 h at 80 °C. The main product of the methanolysis reaction was a de-N-acylated methyl glycoside of sialic acid. N-acetylneuraminic acid oligomers and colominic acid were used to confirm the methanolysis depolymerization efficiency of the α(2→8) saccharides; with the treatment all oligomers produced a common methyl glycoside. For this determination anion exchange chromatography and size exclusion chromatography were both interfaced to an integrated pulsed amperometric detector. Sensitivity and linearity were demonstrated to be sufficient for the application with vaccine dose formulations with low total saccharide concentrations.