Saccharide dosage content of meningococcal polysaccharide conjugate vaccines determined using WHO International Standards for serogroup A, C, W, Y and X polysaccharides.

Potency of meningococcal polysaccharide-protein conjugate vaccines relies on the polysaccharide content to prevent meningitis. NIBSC, as the official national control laboratory in UK, analysed ten different mono- and multi-meningococcal conjugate vaccines, using established International Standards for meningococcal serogroups A, C, W, Y and X, by resorcinol or HPAEC-PAD assay. Most saccharide contents were within ±20% of their claimed content for licensure with taking different O-acetylation levels into consideration, with only MenC content in two vaccines below (by 60% and 54%) the labelled value, however, previous study showed different dosage was not necessarily correlated to the immunogenicity of those vaccines. This study demonstrated the use of International Standards to quantify saccharide content in polysaccharide-based vaccines with different percentage of O-acetylation. These International Standards are suitable to serve as either quantitative standard or calibrator of in-house standards, with supplied stability data.

Collaborative study on saccharide quantification of the Haemophilus influenzae type b component in liquid vaccine presentations.

Before release onto the market, it must be demonstrated that the total and free polysaccharide (poly ribosyl-ribitol-phosphate, PRP) content of Haemophilus influenzae type b (Hib) vaccine complies with requirements. However, manufacturers use different methods to assay PRP content: a national control laboratory must establish and validate the relevant manufacturer methodology before using it to determine PRP content. An international study was organised by the World Health Organization (WHO), in collaboration with the Biological Standardisation Programme (BSP) of the Council of Europe/European Directorate for the Quality of Medicines & HealthCare (EDQM) and of the European Union Commission, to verify the suitability of a single method for determining PRP content in liquid pentavalent vaccines (DTwP-HepB-Hib) containing a whole-cell pertussis component. It consists of HCl hydrolysis followed by chromatographic separation and quantification of ribitol on a CarboPac MA1 column using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The unconjugated, free, PRP is separated from the total PRP using C4 solid-phase extraction cartridges (SPE C4). Ten quality control laboratories performed two independent analyses applying the proposed analytical test protocol to five vaccine samples, including a vaccine lot with sub-potent PRP content and very high free PRP content. Both WHO PRP standard and ribitol reference standard were included as calibrating standards. A significant bias between WHO PRP standard and ribitol reference standard was observed. Study results showed that the proposed analytical method is, in principle, suitable for the intended use provided that a validation is performed as usually expected from quality control laboratories.

Development of a simple and objective evaluation method for thickened liquids using funnels.

Some patients with dysphagia are prone to aspiration of low-viscosity liquids. Thickened liquids are often used in attempts to prevent aspiration. The patients should be given thickened liquids with suitable thickness, and the thickness should be constant at all time. While rotational and cone-and-plate viscometers are used for the evaluation of thickened liquids, they are high-precision and expensive equipment. To control the thickness of liquids, a simple and objective evaluation method is thus necessary. We developed a method to evaluate thickened liquids using funnels, and verified the appropriateness of this method. We measured the outflow times of five thickened liquids through funnels. One of the thickened liquids was a commercially available nutritional supplement, another was made with a thickening agent that contained guar gum, and all others were made with a thickening agent that contained xanthan gum. Four funnels with different stem sizes were tested. We found that the outflow time of thickened liquids through a funnel depended on their viscosities at a shear rate between 10 and 50 s-1 , when the average inner diameter of the stem was in the range of 5.3-9.0 mm, and the volume of the liquid poured into the funnel was 30 mL. The correlation coefficient between the value of the sensory evaluation and the outflow time of the funnel with an average stem ID of 5.3 mm was 0.946. Therefore, this method may be useful in hospital and nursing home kitchens for evaluating thickened liquids. PRACTICAL APPLICATIONS: The findings of this study will help develop a new method for the evaluation of thickened liquids. Funnels made from polypropylene, which are inexpensive and light, were used in this method. The process for measuring the outflow time of thickened liquids through a funnel is simple, and we can obtain quantitative data that are objective. Even though line spread test (LST) is well known as a simple measurement method, nutritional supplements and liquids thickened using a thickening agent containing guar gum have not been evaluated accurately. The funnel method was found to have a stronger correlation with sensory evaluation compared to LST. This method is useful in hospital and nursing home kitchens for evaluating thickened liquids.

Evaluation of candidate international standards for meningococcal serogroups A and X polysaccharide.

Polysaccharide (PS) based meningococcal vaccines are primarily evaluated by physicochemical methods to ensure batches are consistently manufactured. As PS content is determined by different methods across numerous laboratories, there is a need for International Standards (IS) to calibrate the assays. Following the successful introduction of the WHO Meningococcal group C (MenC) IS in 2011, NIBSC initiated projects to prepare similar standards for groups A, W, Y and X (MenA/W/Y/X) to standardise all meningococcal- PS based vaccines. On the basis of results from a collaborative study to evaluate preparations of MenA and MenX PS, both were established by the WHO Expert Committee on Biological Standardization in Oct 2015 as; the First WHO International Standard for the Meningococcal Group A polysaccharide with a content of 0.845 ± 0.043 mg MenA PS per ampoule (expanded uncertainty with coverage factor of k=2.45 corresponding to a 95% level of confidence); the First WHO International Standard for the Meningococcal Group X polysaccharide with a content of 0.776 ± 0.089 mg MenX PS per ampoule (expanded uncertainty with coverage factor of k=2.45), as determined by quantitative NMR. The standards are available from NIBSC, who act as guardians and distributors of the material under the auspices of WHO.

International collaborative study for establishment of the 2nd WHO International Standard for Haemophilus influenzae type b polysaccharide.

In this report we present the results of a collaborative study for the preparation and calibration of a replacement International Standard (IS) for Haemophilus influenzae type b polysaccharide (polyribosyl ribitol phosphate; 5-d-ribitol-(1 â†’ 1)-ß-d-ribose-3-phosphate; PRP). Two candidate preparations were evaluated. Thirteen laboratories from 9 different countries participated in the collaborative study to assess the suitability and determine the PRP content of two candidate standards. On the basis of the results from this study, Candidate 2 (NIBSC code 12/306) has been established as the 2nd WHO IS for PRP by the Expert Committee of Biological Standards of the World Health Organisation with a content of 4.904 ± 0.185mg/ampoule, as determined by the ribose assays carried out by 11 of the participating laboratories.

Evaluation of a candidate International Standard for Meningococcal Group C polysaccharide.

Meningococcal group C (MenC) plain polysaccharide (PS) and conjugate vaccines are primarily evaluated by physicochemical methods to ensure that batches are consistently manufactured. As different assays are employed to quantify the MenC PS content of final formulations and bulk intermediaries, there is a need for an International MenC PS Standard to calibrate internal references used in the different laboratories. Twelve laboratories from nine different countries participated in a collaborative study to determine the MenC PS content of a candidate International Standard MenC PS preparation (08/214) and to assess its suitability. On the basis of the results from this study the candidate standard 08/214 was established as an International Standard for the quantification of MenC PS content in vaccines and components. It has a content of 1.192 ± 0.192 mg MenC PS/ampoule (expanded uncertainty with coverage factor of k = 2.365 corresponding to a 95% level of confidence), as determined by the resorcinol assays carried out by eight of the participating laboratories. The standard is available from The National Institute of Biological Standards and Control who act as guardians and distributors of the material under the auspices of WHO.

A simple and sensitive enzyme immunoassay for determination of soluble type-specific polysaccharide from group B streptococci.

A method was developed for the determination of soluble group B streptococcal type-specific polysaccharide in diluted culture supernatant. The type-specific antigen was immobilized to the solid phase of an enzyme immunoassay using wheat germ agglutinin as a link between the plastic surface and the polysaccharide. The binding of monovalent rabbit antiserum to the type-specific polysaccharide was quantitated by an anti-rabbit IgG-enzyme conjugate. Antisera against each of the polysaccharide types Ia, Ib. II and III gave strong and specific reactions against the respective antigen. The ultimate sensitivity of the assay was 70 pg/ml, as determined for type III polysaccharide. Using this technique, it was found that the concentration of soluble type-specific antigen in a GBS, type III culture supernatant reached a steady-state approximately 3 h after the beginning of the stationary growth phase of the bacteria. Ten type III strains were investigated for synthesis of type-specific polysaccharide, and a positive correlation between the production of capsular and soluble type III antigen was found. There was also an inverse correlation between soluble antigen production and the buoyant densities of these strains. The method described may be used for the serotyping of encapsulated GBS and/or determination of soluble type-specific antigen synthesis.

Measurement of the humoral immune response against Streptococcus pneumoniae type 3 capsular polysaccharide and oligosaccharide containing antigens by ELISA and ELISPOT techniques.

A sensitive ELISA has been developed to study immune responses in mice against Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS) and hexasaccharide (HS)-protein conjugates derived therefrom. An advantage of the described system is that the same microtiter plates can be used for both ELISA and ELISPOT tests with a standardized washing procedure and diluent composition. S3PS induced predominantly IgM antibodies and minute amounts of IgG as measured by ELISA in serum. This was accompanied by large numbers (greater than 14000) of IgM spot-forming cells in the spleen. A shift towards IgG production was achieved by addition of lipid A. HS-protein conjugates induced predominantly IgG antibodies after booster immunization(s). Furthermore these conjugates induced large numbers (greater than 40000) of IgG spot-forming cells (SFC) in the spleen. ELISA and ELISPOT assays on microtiter plates are both reliable and highly reproducible assays for the evaluation of immune responses to S. pneumoniae antigens.

Safety and immunogenicity of high molecular weight polysaccharide vaccine from immunotype 1 Pseudomonas aeruginosa.

The safety and immunogenicity of a high molecular weight polysaccharide from immunotype 1 Pseudomonas aeruginosa were tested in a dose response fashion in adult volunteers. The vaccine lacked toxicity and pyrogenicity for experimental animals. Doses of 50, 75, 150, or 250 microgram were given to groups of individuals as a single dose subcutaneous injection. Doses of 150 and 250 microgram were associated with a significant rise in binding and opsonic antibody at 2 wk postimmunization. Titers remained unchanged for up to 6 mo. The vaccine was almost devoid of toxicity, eliciting no more than a slightly sore and tender arm at the site of injection. High molecular weight polysaccharide antigen appears to induce a good immune response following vaccination that is effective in mediating opsonophagocytic killing of live P. aeruginosa organisms.