Acid hydrolysis conditions for quantification of meningococcal X polysaccharide in a pentavalent vaccine using HPAEC-PAD/ESI-MS.

A selective and sensitive method was evaluated for quantitation of meningococcal X (Men X) polysaccharide in pentavalent meningococcal A, C, W, Y and X conjugate vaccine using different acid hydrolysis conditions like HCl, TFA, HF, HF-TFA, and HF-HCl. High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using CarboPac PA10 column was used to identify the hydrolyzed products based on retention time and its comparison with monosaccharide standards. Complete release of glucosamine (GlcN) from Men X in monovalent bulk and pentavalent vaccine samples was achieved using HF hydrolysis at 80 °C for 2 h. The Men X HF-hydrolyzed polysaccharide to glucosamine along with the reference standard was identified using collision-induced dissociation (CID) electrospray mass spectroscopy and the MS/MS fragments of m/z 162, m/z 144 and m/z 84. Meningococcal polysaccharide concentration was determined with a correlation coefficient r2 >0.99 using polysaccharide reference standard. The serogroups A, W, and Y were converted to their monosaccharides units and quantified using this method however, milder acid hydrolysis 0.1 M HCl 80 °C 2 h for release of sialic acid for Men C polysaccharide was found to be more suitable. These methods will provide necessary tools and prove to be beneficial to laboratories developing new saccharide-based vaccine combinations.

Culture-Confirmed Invasive Meningococcal Disease in Canada, 2010 to 2014: Characterization of Serogroup B Neisseria meningitidis Strains and Their Predicted Coverage by the 4CMenB Vaccine.

The molecular epidemiology of culture-confirmed invasive meningococcal disease (IMD) in Canada from 2010 to 2014 was studied with an emphasis on serogroup B Neisseria meningitidis (MenB) isolates, including their predicted coverage by the 4CMenB vaccine. The mean annual incidence rates of culture confirmed IMD varied from 0.19/100,000 in Ontario to 0.50/100,000 in New Brunswick and 0.59/100,000 in Quebec. In both Quebec and Atlantic region, MenB was significantly more common than other serogroups, while in other provinces, both MenB and serogroup Y (MenY) were almost equally common. The majority of MenB cases (67.0%) were in those aged ≤24 years, while most MenC (75.0%) and MenY (69.6%) cases were in adults more than 24 years old. The 349 MenB isolates were grouped into 103 sequence types (STs), 90 of which belonged to 13 clonal complexes (CCs). A large number of 4CMenB antigen genes were found among the Canadian MenB, which is predicted to encode 50 factor H binding protein (fHbp) types, 40 NHBA types, and 55 PorA genotypes. Provinces and regions were found to have their own unique MenB STs. A meningococcal antigen typing system assay predicted an overall MenB coverage by 4CMenB to be 73.6%, with higher coverage predicted for the two most common STs: 100% for ST154 and 95.9% for ST269, leading to higher coverage in both the Atlantic region and Quebec. Higher coverage (81.4%) was also found for MenB recovered from persons aged 15 to 24 years, followed by strains from infants and children ≤4 years old (75.2%) and those aged 5 to 14 years (75.0%).IMPORTANCE Laboratory surveillance of invasive meningococcal disease (IMD) is important to our understanding of the evolving nature of the Neisseria meningitidis strain types causing the disease and the potential coverage of disease strains by the newly developed vaccines. This study examined the molecular epidemiology of culture-confirmed IMD cases in Canada by examining the strain types and the potential coverage of a newly licensed 4CMenB vaccine on Canadian serogroup B N. meningitidis strains. The strain types identified in different parts of Canada appeared to be unique as well as their predicted coverage by the 4CMenB vaccine. These data were compared to data obtained from previous studies done in Canada and elsewhere globally. For effective control of IMD, laboratory surveillance of this type was found to be essential and useful to understand the dynamic nature of this disease.

Freeze-dried meningococcal vaccine: Total error assessment of a near-infrared method for water content determination.

Accuracy of reference values obtained by the reference method is essential to ensure the quality of a near-infrared (NIR) model. The purpose of this study was to assess the accuracy of a newly developed NIR analytical method for the determination of water content in a freeze-dried meningococcal vaccine by evaluating not only uncertainties associated with the calibration model and NIR measurement procedures but also systematic error arising from the reference Karl-Fischer method. An experimental design based on the standard addition method (SAM) in freeze-dried vaccines is proposed for total error assessment of the NIR analytical system. The accuracy profile showed that the NIR model successfully determined water content over the 1.0-6.7% range. The SAM enabled overall proportional error assessment, which yielded a slope of 1.01. The present study confirmed the suitability of the proposed NIR analytical method for the determination of water content in the freeze-dried meningococcal vaccine. Furthermore, assessment of the overall proportional error proved that the NIR analytical method could be used to accurately estimate differences in water content, therein surpassing the reference method as the method of choice for determining intra- or interbatch variability as well as stability.

La enfermedad meningocócica y las vacunas: algunas respuestas y todavía muchas preguntas / Meningococcal disease and vaccines: still many questions and some answers

No disponible

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.

Quantitation of residual sodium dodecyl sulfate in meningococcal polysaccharide by gas chromatography-mass spectrometry.

Sodium dodecyl sulfate (SDS) is a commonly used surfactant in protein solubilization and also during the polysaccharide purification. A GC-MS method has been developed to quantitate residual SDS in meningococcal polysaccharide serogroups A,C,W,Y and X circumventing the need of spectroscopic assays and HPLC based methods which are either unstable or requires the confirmation by MS. The developed method is based on quantitative conversion of SDS to 1-dodecanol at elevated temperature. Meningococcal polysaccharides and SDS standards were treated with methanolic-HCl and extracted in n-Hexane. The conversion of SDS to 1-dodecanol was confirmed by mass spectra and separation was achieved using a DB-5ms column. The mass spectral analysis of 1-dodecanol showed characteristic ions at m/z 168, 140 and 125. The GC-MS method validation performed on intermediate and purified meningococcal polysaccharides showed linearity with r2 > 0.99 over the concentration range of 2.5-200 µg/ml with LOD and LOQ of 1.27 and 3.85 respectively. The method was found to be precise, robust and accurate with spike recovery ranging 83-117%. The GC-MS method can be used in the quantitation of residual SDS during polysaccharide purification and provides valuable information about consistency of polysaccharide manufacturing process for development of pentavalent meningococcal conjugate vaccine.

Quality by design approach in the development of an ultra-high-performance liquid chromatography method for Bexsero meningococcal group B vaccine.

Bexsero is the first approved vaccine for active immunization of individuals from 2 months of age and older to prevent invasive disease caused by Neisseria meningitidis serogroup B. The active components of the vaccine are Neisseria Heparin Binding Antigen, factor H binding protein, Neisseria adhesin A, produced in Escherichia coli cells by recombinant DNA technology, and Outer Membrane Vesicles (expressing Porin A and Porin B), produced by fermentation of Neisseria meningitidis strain NZ98/254. All the Bexsero active components are adsorbed on aluminum hydroxide and the unadsorbed antigens content is a product critical quality attribute. In this paper the development of a fast, selective and sensitive ultra-high-performance liquid chromatography (UHPLC) method for the determination of the Bexsero antigens in the vaccine supernatant is presented. For the first time in the literature, the Quality by Design (QbD) principles were applied to the development of an analytical method aimed to the quality control of a vaccine product. The UHPLC method was fully developed within the QbD framework, the new paradigm of quality outlined in International Conference on Harmonisation guidelines. Critical method attributes (CMAs) were identified with the capacity factor of Neisseria Heparin Binding Antigen, antigens resolution and peak areas. After a scouting phase, aimed at selecting a suitable and fast UHPLC operative mode for the vaccine antigens separation, risk assessment tools were employed to define the critical method parameters to be considered in the screening phase. Screening designs were applied for investigating at first the effects of vial type and sample concentration, and then the effects of injection volume, column type, organic phase starting concentration, ramp time and temperature. Response Surface Methodology pointed out the presence of several significant interaction effects, and with the support of Monte-Carlo simulations led to map out the design space, at a selected probability level, for the desired CMAs. The selected working conditions gave a complete separation of the antigens in about 5min. Robustness testing was carried out by a multivariate approach and a control strategy was implemented by defining system suitability tests. The method was qualified for the analysis of the Bexsero vaccine.

Impact and effectiveness of meningococcal vaccines: a review / Examen del impacto y la efectividad de las vacunas antimeningocócicas / Impacto e efetividade das vacinas meningocócicas: uma revisão sistemática

ABSTRACT Objectives To summarize and critically evaluate the evidence on the impact and effectiveness of meningococcal vaccination programs around the world in order to inform decisionmaking in Latin America and the Caribbean. Methods A review of the literature was conducted following several components of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. PubMed Central® was searched for papers published in any language from January 1999 - March 2017. Results In all, 32 studies were included, most of which evaluated the meningococcal C conjugate vaccine. Fourteen studies measured effectiveness and 30 measured impact. The effectiveness of polysaccharide vaccines was 65% - 83.7% (different age groups), while the effectiveness of the conjugate vaccines was 66% - 100%. Incidence decline of laboratory-confirmed meningococcal disease for the conjugate vaccine ranged from 77% - 100% among different ages groups. The only study that evaluated the protein subunit vaccine reported a vaccine effectiveness of 82.9%. Conclusions The studies reviewed show impact and effectiveness of both polysaccharide vaccines and conjugate vaccines on vaccine-serogroup meningococcal disease. The conjugate vaccines, however, show higher impact and effectiveness with longer-lasting protection over the polysaccharide vaccines. Given the variance in potential use of a meningococcal vaccine, epidemiological surveillance systems should be strengthened to inform national decisions.

RESUMEN Objetivos Resumir y evaluar críticamente los datos científicos sobre la repercusión y la eficacia de los programas de vacunación antimeningocócica en todo el mundo para orientar la toma de decisiones en América Latina y el Caribe. Métodos Se realizó un examen de la bibliografía siguiendo varios componentes de las directrices correspondientes a los elementos de notificación preferidos para revisiones sistemáticas y metanálisis. Se hicieron búsquedas en PubMed Central® para encontrar documentos publicados en cualquier idioma desde enero de 1999 hasta marzo del 2017. Resultados En total, se incluyeron 32 estudios, en cuya mayoría se evaluaba la vacuna antimeningocócica conjugada contra el serotipo C. En 14 estudios se midió la efectividad y en 30, el impacto. La efectividad de las vacunas polisacarídeas se encontraba entre 65% y 83,7% (grupos etarios diferentes), mientras que la de las vacunas conjugadas, entre 66% y 100%. Gracias a la vacuna conjugada, la disminución de la incidencia de la enfermedad meningocócica confirmada en laboratorio varió entre 77% y 100% en diferentes grupos etarios. En el único estudio en el que se evaluó la vacuna de subunidades proteínicas se notificó que su efectividad era de 82,9%. Conclusiones Los estudios examinados muestran el impacto y la efectividad tanto de las vacunas polisacarídeas como de las vacunas conjugadas en relación con la enfermedad meningocócica causada por los serotipos utilizados en las vacunas. Sin embargo, las vacunas conjugadas demuestran que tienen un mayor impacto y efectividad que las vacunas polisacarídeas para dar una protección más duradera. En vista de la varianza del posible uso de una vacuna antimeningocócica, se deben fortalecer los sistemas de vigilancia epidemiológica para orientar las decisiones nacionales.

RESUMO Objetivos Sumarizar e analisar de maneira crítica as evidências sobre o impacto e a efetividade dos programas de vacinação contra doença meningocócica em todo o mundo para subsidiar a tomada de decisão na América Latina e no Caribe. Métodos Uma revisão da literatura científica foi realizada de acordo com vários componentes das diretrizes dos Principais itens para relatar revisões sistemáticas e meta-análises (PRISMA). Foi feita uma pesquisa da base de dados PubMed Central® em busca de estudos publicados em qualquer idioma de janeiro de 1999 a março de 2017. Resultados Trinta e dois estudos foram selecionados, sendo que a maioria avaliou a vacina conjugada contra o meningococo C. Quatorze artigos avaliaram a efetividade da vacina e 30, o impacto. A efetividade das vacinas polissacarídicas foi de 65% a 83,7% (em diferentes faixas etárias) e a das vacinas conjugadas foi de 66% a 100%. A redução com o uso da vacina conjugada da incidência de doença meningocócica confirmada por laboratório variou de 77% a 100% em diferentes faixas etárias. O único estudo que avaliou a vacina de subunidade proteica informou uma efetividade de 82,9%. Conclusões Os estudos examinados indicam impacto e efetividade da vacina polissacarídica e da vacina conjugada para doença meningocócica do respectivo sorogrupo vacinal. As vacinas conjugadas demonstram maior impacto e efetividade e proteção mais duradoura em relação às vacinas polissacarídicas. Diante da variação do uso em potencial da vacina meningocócica, os sistemas de vigilância epidemiológica devem ser reforçados visando subsidiar a tomada de decisão das autoridades nacionais.

Robust Assessment of Molecular Size Distribution of Meningococcal AC Vaccine.

The molecular size of meningococcal polysaccharides is an important physicochemical parameter that is related to immunogenicity and efficacy. A simple method for size-exclusion chromatography was developed, optimized, and applied for safe and rapid fractionation of meningococcal polysaccharide AC vaccine. Pooling of the fractions collected from size-exclusion chromatography was investigated and evaluated, rather than analyzing each fraction separately, for determining the percentages of meningococcal polysaccharide A and C that were eluted before the distribution coefficient of 0.5. Pooling is preferred rather than analyzing each fraction individually, as it is easily handled, faster, simpler, less expensive, more accurate, safe, and applicable. The developed method was validated and successfully applied for the determination of meningococcal polysaccharide vaccine serotype A and C in quality-control and commercial samples.

Quantification by LC-MS(E) of outer membrane vesicle proteins of the Bexsero® vaccine.

Meningococcal disease is a major cause of morbidity and mortality worldwide. Its epidemiology is currently dominated by five capsular serogroups (A, B, C, W, and Y). While effective vaccines already exist for serogroups A, C, W and Y, except for under clonal outbreaks, no vaccine was available against serogroup B. Recently, a four component vaccine, Bexsero(®), designed to prevent infection caused by this serogroup, has been approved in Europe and other Countries for use in individuals from two months of age and older. The active components of this vaccine are three recombinant proteins identified by reverse vaccinology combined with detergent extracted outer membrane vesicles (DOMV) prepared from a New Zealand epidemic strain. Considering their intrinsic complexity, we performed additional characterization of DOMVs on top of the standard quality control testing carried out for batch release. We applied the Hi3 label-free LC-MS(E) methodology to qualitatively and quantitatively characterize the DOMV protein content. We first, successfully investigated the robustness and the accuracy of the methodology for the DOMV characterization and we then applied it to compare six DOMV production lots. Around 100 proteins were quantified from each preparation. When classified according to their predicted cellular localization, about 90% of the total protein amount belongs consistently to the outer membrane compartment. Using nonparametric hypothesis testing and complementary log-log linear regression, the quantifications of a subset of 21 proteins common to all lots and including approximately 90% (85-92%) of the total protein amount quantified in any DOMV lot were found consistent across lots. The relevance of these results is two-fold, showing that the Hi3 quantification methodology is robust for a broad range of proteins and indicating that the manufacturing process currently used for the production of the Bexsero(®) DOMV components is highly reproducible and consistent.

Efetividade da vacina meningocócica C conjugada e caracterização da neisseria miningitidis em Salvador Bahia / Effectiveness of meningococcal C conjugate vaccine and characterization of Neisseria meningitidis in Salvador, Bahia

Introdução: A doença meningocócica (DM) é causada pela bactéria Neisseria meningitidis, sendo um importante problema de saúde pública no mundo. Atualmente, a Neisseria meningitidis sorogrupo C (NmC) tem sido o principal agente da DM na Bahia. Em 2010 ocorreu uma epidemia de DM pela NmC em Salvador, e a fim de contê-la, a Secretaria Estadual de Saúde introduziu em fevereiro de 2010, a vacina meningocócica C conjugada (MenC) para crianças menores de cinco anos, incluindo campanhas de vacinação para indivíduos de 10 a 24 anos. Objetivos: Descrever a incidência da DM, avaliar a efetividade da vacina MenC e caracterizar os fenótipos e genótipos das cepas circulantes da N. meningitidis nos períodos pré e pós-introdução da vacina MenC. Metodologia: Realizamos um estudo descritivo-analítico, comparando incidências nas coortes de vacinados e não vacinados nos períodos pré e pós-introdução da vacina MenC. Analisamos a efetividade da vacina MenC utilizando o método “screening” e um estudo tipo caso-controle. A efetividade da vacina MenC foi baseada no odds-ratio (IC 95%; pvalor <0,05). Para caracterização molecular da NmC, utilizamos a técnica de Eletroforese em Campo Pulsátil (PFGE) e da Tipagem de Sequências Multilocus (MLST)...

Introduction: Meningococcal disease (MD) is caused by bacterium Neisseria meningitidis and is a major public health problem worldwide. Currently the Neisseria meningitidis serogroup C (NmC) has been the main cause of MD in Bahia, Brazil. In order to contain the 2010 epidemic of MD caused by NmC that occurred in the city of Salvador, the State Department of Health introduced in February 2010 the meningococcal C conjugate vaccine (MenC) to <5 year-old children, including vaccination campaigns for individuals from 10-24 years. Objectives: Describe trends in incidence of MD, estimate the effectiveness of MenC vaccine, and characterize the phenotypes and genotypes of the circulating strains of N. meningitidis in the pre and post-introduction of the MenC vaccine. Methods: A descriptive analytical study was realized comparing incidences in cohorts vaccinated and unvaccinated pre and post introduction of the MenC vaccine. We analyze the effectiveness of MenC vaccine using the screening method and a case-control study. The effectiveness of MenC vaccine was based on the odds-ratio (CI 95%). We performed molecular analyses by pulsed field gel electrophoresis (PFGE) and by multi-locus sequencing typing (MLST)...

Serogroup quantitation of multivalent polysaccharide and polysaccharide-conjugate meningococcal vaccines from China.

The active components of most meningococcal vaccines are four antigenic serogroup capsular polysaccharides (A, C, Y, W135). The vaccines, monovalent or multivalent mixtures of either free polysaccharides or polysaccharides conjugated to antigenic carrier proteins, may be in liquid or lyophilised formulations, with or without excipients. Acid hydrolysis and chromatographic methods for serogroup quantitation, which were previously optimised and qualified using polysaccharide-based standards and a narrow range of real vaccines, are here challenged with multiple lots of a broad assortment of additional multivalent polysaccharide-based meningococcal vaccine products. Centrifugal filtration successfully removed all interfering lactose excipient without loss of polysaccharides to allow for the determination of Y and W135 serogroups. Replicate operations by three different analysts indicated high method reproducibility. Results indicated some lot-to-lot and product-to-product variations. However, all vaccines were within general specifications for each serogroup polysaccharide, with the exception of all lots of one polysaccharide vaccine - which by these methods were found to be deficient in the serogroup A component only. These robust techniques are very useful for the evaluation of antigen content and consistency of manufacture. The deformulation, hydrolysis and chromatographic methods may be adaptable for the evaluation of other types of polysaccharide-based vaccines.

Quantitative proteomics reveals distinct differences in the protein content of outer membrane vesicle vaccines.

At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in more detail, the protein content of detergent-extracted OMV is compared with two detergent-free alternatives. A novel proteomics strategy has been developed that allows quantitative analysis of many biological replicates despite inherent multiplex restrictions of dimethyl labeling. This enables robust statistical analysis of relative protein abundance. The comparison with detergent-extracted OMV reveales that detergent-free OMV are enriched with membrane (lipo)proteins and contain less cytoplasmic proteins due to a milder purification process. These distinct protein profiles are substantiated with serum blot proteomics, confirming enrichment with immunogenic proteins in both detergent-free alternatives. Therefore, the immunogenic protein content of OMV vaccines depends at least partially on the purification process. This study demonstrates that detergent-free OMV have a preferred composition.

Flow cytometry: an alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant.

Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r(2)) were routinely achieved for a broad range of antigen doses from 0 to 150 µg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen-adjuvant desorption methods.

Bioanalysis of meningococcal vaccines.

Meningococcal meningitis is feared because of the rapid onset of severe disease from mild symptoms and, therefore, is an important target for vaccine research. Five serogroups, defined by the structures of their capsular polysaccharides, are responsible for the vast majority of disease. Protection against four of these five serogroups can be obtained with polysaccharide or glycoconjugate vaccines, in which fragments of the capsular polysaccharides attached to a carrier protein generate anticarbohydrate immune responses, whilst protection against group B disease requires protein immunogens, often presented in vesicles containing outer membrane proteins. Glycoconjugate vaccines are now an established technology, but outer-membrane protein vaccines are still under development and present significant challenges. This review discusses physicochemical approaches to the characterization and quality control of these vaccines, as well as highlighting the problems and differences in vaccine design required for protection against different serogroups of the same species of pathogen.

La técnica de Western Blot aplicada a la vacuna antimeningocócica VA MENGOC BC® / Western Blot technique applied to VA MENCOG BC® antimeningococcal vaccine

Se desarrolló y validó la técnica de Western Blot aplicada a la vacuna antimeningocócica VA MENGOC BC® producida en el Instituto Finlay con el objetivo de demostrar criterio de identidad. Con el empleo de esta técnica se identificaron las proteínas antigénicas del tipo P1, P3, P5, 70 y 80 K presentes en la vesícula de membrana externa y vacuna final, por lo cual se utilizó como antisuero la gamma antimeningocócica. Los parßmetros desarrollados en la validación de la técnica fueron: especificidad, límite de detección, repetibilidad, precisión intermedia, reproducibilidad y robustez. La técnica de identidad cumplió con los parámetros se±alados anteriormente, por lo que se considera validada.

ABSTRACT Western Blot technique applied to VA MENGOC BC® antimeningococcal vaccine was developed and validated and produced in "Carlos J. Finlay" Institute to demonstrate the identity criterion. Using this technique it was possible to identify antigenic proteins type P1, P3, P5 70 and 80 K present in the vesicle of external membrane and final vaccine, thus, we used the antimeningococcal gamma. Parameters developed in validation of this technique included: specificity, detection limit, repetition, average accuracy, reproduction, and strength, identity technique fulfilled with abovementioned parameters, considering like validated.

La técnica de Western Blot aplicada a la vacuna antimeningocócica VA MENGOC BC® / Western Blot technique applied to VA MENCOG BC® antimeningococcal vaccine

Se desarrolló y validó la técnica de Western Blot aplicada a la vacuna antimeningocócica VA MENGOC BC® producida en el Instituto Finlay con el objetivo de demostrar criterio de identidad. Con el empleo de esta técnica se identificaron las proteínas antigénicas del tipo P1, P3, P5, 70 y 80 K presentes en la vesícula de membrana externa y vacuna final, por lo cual se utilizó como antisuero la gamma antimeningocócica. Los parßmetros desarrollados en la validación de la técnica fueron: especificidad, límite de detección, repetibilidad, precisión intermedia, reproducibilidad y robustez. La técnica de identidad cumplió con los parámetros se±alados anteriormente, por lo que se considera validada(AU)

ABSTRACT Western Blot technique applied to VA MENGOC BC® antimeningococcal vaccine was developed and validated and produced in "Carlos J. Finlay" Institute to demonstrate the identity criterion. Using this technique it was possible to identify antigenic proteins type P1, P3, P5 70 and 80 K present in the vesicle of external membrane and final vaccine, thus, we used the antimeningococcal gamma. Parameters developed in validation of this technique included: specificity, detection limit, repetition, average accuracy, reproduction, and strength, identity technique fulfilled with abovementioned parameters, considering like validated(AU)

Purificación de lipopolisacárido de Neisseria meningitidis a partir de unafracción colateral del proceso de producción de VA-MENGOC-BC® / Purification of Neisseria meningitidis lipopolysaccharide starting from a collateral fraction of the process of the antimeningococcal vaccine, VA-MENGOC-BC production process

El trabajo tuvo como objetivo purificar lipopolisacáridos (LPS) de Neisseria meningitidis a partir de una fraccióncolateral del proceso de producción de la vacuna antimeningocócica VA-MENGOC-BC®, el sobrenadante que seobtiene del paso de ultracentrifugación durante el proceso de extracción de las proteínas de la membrana externa delmeningococo. La purificación se realizó mediante precipitación con etanol al 80 por ciento, extracción de las proteínas con fenol al 90 porciento entre 65-70 ºC y ultracentrifugación fraccionada a 105,000 g. Se obtuvieron tres lotes de LPS, en total 1,069 g, con un contenido de proteínas, ácidos nucleicos y ácido sálico respecto al LPS de 0,5 por ciento, 0,3 por ciento y 2,2 por ciento (m/m),respectivamente. La evaluación por cromatografía mostró una alta integridad molecular, con valores de constante de distribución reproducibles (0,36-0,38) y una posible asociación del ácido siálico al LPS. Se apreció homogeneidad en el perfil electroforético de los tres lotes y alta actividad endotóxica. El LPS purificado fue identificado fundamentalmente como del inmunotipo L3,7,9. El procedimiento de purificación empleado permite aprovechar una fracción colateral del proceso de producción de la vacuna, es escalable, no incluye métodos cromatográficos, y posibilita la obtención de gran cantidad de LPS de Neisseria meningitidis, no disponible en el mercado, con elevada pureza y alta actividad endotóxica(AU)

The work aimed at purifying lipopolysaccharides (LPS) of Neisseria meningitidis from a collateral fraction of the antimeningococcal BC vaccine, VAMENGOC-BC®. production process, the supernatant obtained from the ultra centrifugation stage during the proteins extraction process of the meningococcus outer membrane. The purification was carried out by precipitation 80 percent ethanol, protein extraction with 90 percent phenol from 65-70 ºC and fractional ultra centrifugation at 105.000 g. Three lots of LPS were obtained, in total 1.069 g, with a content of proteins, nucleic acids and sialic acid in respect to the LPS of 0.5 percent, 0.3 percent and 2.2percent (m/m) respectively. The assessment by chromatography showed a high molecular integrity. with constant valves of reproducible distribution (Kd 0.36 0.38) and a possible sialic acid association to the LPS. Homogeneitywas observed in the electrophoretic profile of the three lots and a high endotoxic activity. The purified LPS was mainly identified as the inmunotype L3,7,9. The purification procedure used allows making use of a collateral fraction of the vaccine production process, it is scalable, it does not include chromatographic methods and makes easy the obtainment of large quantityof LPS of Neisseria meningitidis, wihich it is non available on the market, with high purity and high endotoxic activity(AU)

Vacunas antimeningocócicas / Vaccines against Neisseria meningitidisg

Se revisa la situación actual de las vacunasantimeningocócicas. Por una parte, las vacunaspolisacáridas simples frente a los serogrupos A, C, Y y W-135 y, por otra, las polisacáridas conjugadas a distintos transportadores proteicos, monovalentes o multivalentes.Respecto a las vacunas frente a Neisseria meningitidisserogrupo B, se analizan las experiencias basadas en lasproteínas de membrana externa y su uso en el control debrotes epidémicos, y las expectativas de una vacunauniversal que se podrían alcanzar con las técnicas devacunología inversa

The present article reviews the state of the art on vaccines against Neisseria meningitidis, from plain polysaccharide to mono- and multivalent protein-polysaccharide conjugate vaccines targeting A, C, Y and W-135 serogroups. We also review immunization againstserogroup B Neisseria meningitidis using protein-basedvaccines composed of outer membrane vesicles and theiruse in the control of epidemic outbreaks, as well asexpectations of a universal vaccine, which could beachieved with reverse vaccinology techniques (AU)

Vacunas combinadas / Combined vaccines

Las vacunas combinadas son las que contienen antígenosque pertenecen a 2 o más microorganismos; se conocenhace ya 60 años, desde que en 1948 se autorizó la vacunaDTP. La disponibilidad de vacunas combinadas facilitaaumentar el número de antígenos en los calendarios deinmunizaciones sistemáticas, con un menor número deinyecciones y una mayor aceptación por parte del personalsanitario y de la población, y permite obtener mejorescoberturas vacunales. A pesar del importante aumento delnúmero de vacunas sistemáticas administradas a los niños(en los últimos 100 años se ha pasado de 1 a 14 vacunas),la cantidad de componentes antigénicos no sólo no haaumentado, sino que ha disminuido (de 200 en 1903, a 130en 2007), al obtenerse vacunas cada vez más purificadas.El aumento creciente del número de vacunas en loscalendarios de inmunizaciones en los últimos años hahecho progresar la investigación y el desarrollo de vacunas combinadas. A partir de la vacuna trivalente DTP, en las formas DTPe y DTPa, se han desarrollado 2 grupos de vacunas combinadas, y en la actualidad se dispone de tetravalentes, pentavalentes y una hexavalente con DTPa.Hay un tercer grupo de vacunas combinadas en cuyacomposición no está la vacuna DTP. Finalmente, hay quedestacar una vacuna heptavalente, en la que se añade auna de las pentavalentes (DTPe-HB-Hib) las vacunasconjugadas para el meningococo A y C; esta vacuna se hapresentado en marzo de 2007 a la Agencia Europea deMedicamentos para los países del «cinturón de lameningitis»

Combined vaccines contain antigens belonging to two ormore microorganisms; these vaccines have existed for thelast 60 years, ever since the diphtheria-tetanus-pertussis(DTP) vaccine was authorized in 1948. The availability of combined vaccines helps to increase the number of antigens in immunization schedules, with a lower number of injections and greater acceptance by health workers and the general population. Furthermore, these vaccines improve vaccination coverage. Despite the substantial increase in the number of systematic vaccinesadministered to children (increasing in the last 100 years from 1 to 14 vaccines), the quantity of antigeniccomponents has not only not increased but has actuallydecreased (from 200 and 1903 to 130 in 2007) due to theavailability of increasingly purified vaccines. The upward trend in the number of vaccine immunization schedules in the last few years has stimulated research and development into combined vaccines. Based on thetrivalent DTP vaccine, either whole-cell DTP or DTaP, twogroups of combined vaccines have been developed andtetravalent, pentavalent and hexavalent forms arecurrently available with DTPa. There is a third group ofcombined vaccines that does not contain the DTP vaccine.Finally, there is a heptavalent vaccine in which theconjugate vaccines for meningococcus A and C are addedto one of the pentavalent vaccines (whole cell DTP-HBHib); this vaccine was presented in March 2007 to theEuropean Medicines Agency for countries in the meningitisbelt