Molecular size distribution assessment of Haemophilus influenzae vaccine containing lactose by HPAEC-PAD and colorimetric assays.

Molecular size distribution of Haemophilus influenzae type b (Hib) conjugate vaccine is an important indicator for its immunogenicity and stability. Molecular size distribution was evaluated by High-Performance Protein Chromatography on Sepharose CL-4B column, and fractions were pooled. The use of high flow rate, incorporation of a calibration standard with the injected buffer and pooling method yielded a superior assay compared to conventional pharmacopeial method. The pools were analyzed for determination of distribution coefficient (KD) of 0.2 and 0.5 using two validated techniques: High Performance Anion Exchange Chromatography with pulsed amperometric detection (HPAEC-PAD) for ribitol determination and an optimized colorimetric assay for phosphorus determination. Linearity was achieved over range of 0.10-10.0 µg/mL and 1.0-8.0 µg/mL with LOD of 0.03 and 0.28 µg/mL for HPAEC and colorimetric assays, respectively. The developed assays were successfully applied in quality control monitoring of Hib conjugate vaccine. The optimized colorimetric method had shortened the analysis time to 25 min compared to 3.5 h for the European pharmacopeial assay by modifying the burning process. HPAEC stability results revealed 40% decrease in MSD after stressed storage conditions. The proposed assays offer a reliable and economic platform for monitoring the quality attributes of Hib for biopharma industry.

Use of NMR as an analytical tool in the process development of conjugate vaccines against Haemophilus influenzae type b (Hib) and meningococcal serogroup A (MenA).

The native structure of the bacterial polysaccharide is the key immunogenic component of conjugate vaccines and antibodies raised against the polysaccharide structure are responsible for providing protection against the corresponding pathogen. The manufacturing process of conjugate vaccines is very complex and has various biological and chemical steps. It is important to monitor the process to ensure that the structural identity of the polysaccharide is maintained throughout the process. NMR spectroscopy can be used as a versatile analytical tool to monitor the structural integrity of the polysaccharide component from isolated polysaccharide to conjugate vaccine and for identifying different impurities generated during the process.

Vacunación frente a la gripe estacional en las personas mayores. Evaluación de la vacuna tetravalente. Informe de posicionamie / Seasonal flu vaccination for older people: Evaluation of the quadrivalent vaccine. Positioning repor

La gripe es un importante problema de salud pública, particularmente en las personas susceptibles de presentar complicaciones asociadas, personas mayores, niños menores de 2 años, enfermos crónicos, inmunocomprometidos y embarazadas. Pero, además, la gripe tiene un gran impacto sanitario con un aumento de la demanda asistencial y un espectacular aumento de las visitas ambulatorias, sobrecargando los servicios de urgencias y hospitalarios. Durante los brotes epidémicos, las tasas de hospitalización de las personas mayores de 65 años son máximas y la mortalidad notificada por gripe en la temporada 2017/2018 ha sido de 960 defunciones. La vacunación antigripal estacional es el método con una mayor relación coste-efectividad de prevención primaria de la gripe, reduciendo las enfermedades respiratorias relacionadas, el número de visitas a las consultas médicas, el número de hospitalizaciones y muertes en personas de alto riesgo y el absentismo laboral en adultos. En los últimos años la gripe B ha recibido escasa atención en la literatura científica y, sin embargo, en períodos interepidémicos, la gripe B puede ser una de las principales causas de epidemias de gripe estacional, causando una considerable morbimortalidad y un aumento de costes. La vacuna tetravalente, a diferencia de la trivalente, obtiene una protección inmunológica frente al segundo linaje de la gripe B y, de acuerdo con una revisión crítica de la literatura científica, proporciona una protección más amplia sin afectar a la inmunogenicidad de las otras 3 cepas vacunales comunes a las vacunas trivalente y tetravalente. La vacuna tetravalente es coste-efectiva al disminuir el número de casos de gripe y siempre es una intervención rentable, con un importante ahorro de coste para el sistema de salud y para la sociedad, disminuyendo las tasas de hospitalización y de mortalidad asociadas a las complicaciones de la gripe

Influenza is a significant health problem, particularly in those persons susceptible to having associated complications, older people, children less than 2 years, patients with chronic diseases, immunocompromised patients, and pregnant women. But influenza also has a large impact on the health system, with an increase in the healthcare demand and a spectacular increase in outpatient visits, overloading the emergency and hospital services. During epidemic outbreaks, the hospital admission rates of people over 65 years are at a maximum, and the mortality notified for the 2017/2018 influenza season was 960 deaths. The seasonal anti-influenza vaccine is the method with a better cost-effective ratio of primary prevention of influenza, reducing associated respiratory diseases, the number of hospital admissions, and deaths in high risk individuals, as well as work absenteeism in adults. In the last few years, influenza B has received little attention in the scientific literature, although in the periods between epidemics influenza B can be one of the main causes of seasonal epidemics, causing considerable morbidity and mortality and an increase in costs. The quadrivalent vaccine has a second-line immunological protection against influenza B, and according to a critical review of the scientific literature, it provides wider protection without affecting immunogenicity of the other three vaccine strains common to the trivalent and tetravalent vaccine. The quadrivalent vaccine is cost-effective in reducing the number of influenza cases, and is always a worthwhile intervention, with a significant cost saving for the health system and for society, by reducing the hospital admission rates and mortality associated with the complications of influenza

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.

Process development of a New Haemophilus influenzae type b conjugate vaccine and the use of mathematical modeling to identify process optimization possibilities.

Vaccination is one of the most successful public health interventions being a cost-effective tool in preventing deaths among young children. The earliest vaccines were developed following empirical methods, creating vaccines by trial and error. New process development tools, for example mathematical modeling, as well as new regulatory initiatives requiring better understanding of both the product and the process are being applied to well-characterized biopharmaceuticals (for example recombinant proteins). The vaccine industry is still running behind in comparison to these industries. A production process for a new Haemophilus influenzae type b (Hib) conjugate vaccine, including related quality control (QC) tests, was developed and transferred to a number of emerging vaccine manufacturers. This contributed to a sustainable global supply of affordable Hib conjugate vaccines, as illustrated by the market launch of the first Hib vaccine based on this technology in 2007 and concomitant price reduction of Hib vaccines. This paper describes the development approach followed for this Hib conjugate vaccine as well as the mathematical modeling tool applied recently in order to indicate options for further improvements of the initial Hib process. The strategy followed during the process development of this Hib conjugate vaccine was a targeted and integrated approach based on prior knowledge and experience with similar products using multi-disciplinary expertise. Mathematical modeling was used to develop a predictive model for the initial Hib process (the 'baseline' model) as well as an 'optimized' model, by proposing a number of process changes which could lead to further reduction in price. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:568-580, 2016.

Dual orientation of the outer membrane lipoprotein P6 of nontypeable haemophilus influenzae.

The majority of outer membrane (OM) lipoproteins in Gram-negative bacteria are tethered to the membrane via an attached lipid moiety and oriented facing in toward the periplasmic space; a few lipoproteins have been shown to be surface exposed. The outer membrane lipoprotein P6 from the Gram-negative pathogenic bacterium nontypeable Haemophilus influenzae (NTHi) is surface exposed and a leading vaccine candidate for prevention of NTHi infections. However, we recently found that P6 is not a transmembrane protein as previously thought (L. V. Michel, B. Kalmeta, M. McCreary, J. Snyder, P. Craig, M. E. Pichichero, Vaccine 29:1624-1627, 2011). Here we pursued studies to show that P6 has a dual orientation, existing infrequently as surface exposed and predominantly as internally oriented toward the periplasmic space. Flow cytometry using three monoclonal antibodies with specificity for P6 showed surface staining of whole NTHi cells. Confocal microscopy imaging confirmed that antibodies targeted surface-exposed P6 of intact NTHi cells and not internal P6 in membrane-compromised or dead cells. Western blots of two wild-type NTHi strains and a mutant NTHi strain that does not express P6 showed that P6 antibodies do not detect a promiscuous epitope on NTHi. Depletion of targets to nonlipidated P6 significantly decreased bactericidal activity of human serum. Protease digestion of surface-exposed P6 demonstrated that P6 is predominantly internally localized in a manner similar to its homologue Pal in Escherichia coli. We conclude that P6 of NTHi is likely inserted into the OM in two distinct orientations, with the predominant orientation facing in toward the periplasm.

Development of a high-resolution magic-angle spinning nuclear magnetic resonance identity assay of the capsular polysaccharide from Haemophilus influenzae type b present in cetavlon precipitate.

We describe the use of high-resolution magic-angle spinning nuclear magnetic resonance to control the identity of the capsular polysaccharide from Haemophilus influenzae type b (Hib) present in the cetavlon precipitate. This step is one of the earliest in the purification of this polysaccharide, which is further used in the production of Hib polysaccharide-protein conjugate vaccine. The effects of sample procedure and magnetic field strength have been investigated. Since this assay is rapid and simple, it may represent a useful technique for characterization of polysaccharides present in complex and insoluble matrices. Moreover, it allows a rapid evaluation of the structure of the produced polysaccharides very early on during the production process and is as such an essential analytical tool before starting the purification process.

[FMRP immunodetection on hair roots: application to the diagnosis of fragile X syndrome]. / Estudio de la proteína FMRP en la raíz de cabello: aplicación al diagnóstico del síndrome del cromosoma X frágil.

INTRODUCTION: Fragile X syndrome is the most common inherited form of mental retardation. The absence of FMRP protein, codified by the FMR1 gene, results in fragile X phenotype. DNA-based diagnostic methods determine the length of the CGG repeat within the FMR1 gene, the main mutation causing the syndrome. Immunohistochemical diagnostic tests detect all mutations leading to the absence of FMRP expression. Results of the antibody test on hair roots correlate with intellectual quotient in affected men and women. PATIENTS AND METHODS: Immunohistochemical techniques were used to study FMRP expression in hair roots in a control group to establish the correlation with the length of the CGG repeat. Subsequently, 65 girls and boys with mental retardation attending special schools were screened by using the FMRP test on hair roots. RESULTS: Males and females molecularly characterized as within the normal and premutated range expressed FMRP in more than 70 % of hair roots. Full mutation carriers expressed FMRP in less than 70 % of hair roots. Immunohistochemical studies in males and females with mental retardation led to the identification of one affected male. CONCLUSIONS: Fragile X syndrome detection by immunohistochemical testing of hair roots is a valid method of population screening because of the relative noninvasiveness of obtaining samples, and the ease and rapidness of the technique, which can be applied to routine clinical practice.

Development of an in vitro potency test for tetanus vaccines: an immunoassay based on Hc fragment determination.

In the three Rs rule context, we have developed a method to quantify tetanus toxoid by ELISA detection of the specific Hc fragment. This fragment and several anti Hc fragment antibodies have been described as protective in mice models. By developing this assay, we have detected Hc presence in tetanus toxoid. Therefore, this functional in vitro assay could replace in vivo potency assays. We have evaluated the analytical performances of this ELISA in specificity, sensitivity, precision, and linearity tests on different combined vaccine formulations. This in vitro assay has been applied to various multi-component vaccines. Results are expressed in Lf Hc/ml with reference to a purified tetanus toxoid standard expressed in Lf/ml.

Application of genomics and proteomics for identification of bacterial gene products as potential vaccine candidates.

The ability of bioinformatics to characterize genomic sequences from pathogenic bacteria for prediction of genes that may encode vaccine candidates, e.g. surface localized proteins, has been evaluated. By applying appropriate tools for genomic mining to the published sequence of Haemophilus influenzae Rd genome, it was possible to identify a putative vaccine candidate, the outer membrane lipoprotein, P6. Proteomics complements genomics by offering abilities to rapidly identify the products of predicted genes, e.g. proteins in outer membrane preparations. The ability to identify the P6 protein uniquely from entries in a sequence database from the expected peptide-mass fingerprint of P6 demonstrates the power of proteomics. The application of proteomics for identification of vaccine candidates for another pathogenic bacterium, Helicobacter pylori using two different approaches is described. The first involves rapid identification of a series of monoclonal antibody reactive proteins from N-terminal sequence tags. The other approach involves identification of proteins in outer membrane preparations by 2-D electrophoresis followed by trypsin digestion and peptide mass map analysis. Our combined studies demonstrate that utilization of genome sequences by application of bioinformatics through genomics and proteomics can expedite the vaccine discovery process by rapidly providing a set of potential candidates for further testing.

Quantitative determination of saccharide in Haemophilus influenzae type b glycoconjugate vaccines, alone and in combination with DPT, by use of high-performance anion-exchange chromatography with pulsed amperometric detection.

The stability and integrity of glycoconjugate vaccines requires determination of the total saccharide and quantification of the unbound or free saccharide present. The traditional assay for Hib conjugates, based on colorimetric determination of ribose, has been much improved by the use of base hydrolysis and analysis of the Hib subunit generated using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The production of this subunit was confirmed by NMR analysis. However, quantification of free Hib saccharide using this method was not possible in the combination vaccines evaluated due to interferences emanating from DPT. Thus a method based on TFA hydrolysis followed by the chromatographic separation and quantification of ribitol on a CarboPac MA1 column was developed. The method is selective, and with the use of ED40 electrode, requires only nanomole amounts for the chromatographic step, thereby ensuring that free saccharide can be monitored accurately in the formulated Hib-CRM vaccine alone and when in combination with other vaccines.

Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines.

We recently described the use of ion exchange chromatography for analysis and the industrial scale preparation of pools of oligosaccharides of intermediate chain length from polysaccharides of Haemophilus influenzae type b (Hib) and Neisseria meningitidis groups A and C. These negatively charged "sized" oligosaccharides are activated and conjugated to the carrier protein (CRM197) to prepare the corresponding glycoconjugate vaccines. Characterization and accurate determination of the degree of polymerization (DP) of the pool of oligosaccharides is essential for the consistent production of these conjugate vaccines. This paper describes the colorimetric assays used for determination of the average DP of the Hib and meningococcal oligosaccharides, and the qualification of these assays achieved by size characterization of the respective oligosaccharides by use of physicochemical methods, including liquid chromatography, mass spectrometry (ionspray) and NMR spectroscopy.

Molecular size characterization of Haemophilus influenzae type b polysaccharide-protein conjugate vaccines.

Current vaccines against Haemophilus influenzae type b (Hib) consist of capsular polysaccharide, polyribosylribitol phosphate (PRP), chemically conjugated to a carrier protein. The stability of the conjugate is essential for vaccine efficacy, as the target population for this vaccine includes infants, who do not mount an immune response to free polysaccharide vaccines. A method has been developed for determining structural stability and batch-to-batch consistency of Hib vaccines by the application of fast protein liquid chromatography (FPLC). This FPLC method is fast, reproducible, and can be used to evaluate single human doses of Hib vaccines. We have shown that the FPLC elution profiles provide a suitable indicator of vaccine stability under normal and degradative conditions. The method may also be applicable to other conjugate vaccines such as meningococcal and pneumococcal protein-polysaccharide conjugates.