Demyelination Lesions Do Not Correlate with Clinical Manifestations by Bordetella pertussis Toxin Concentrations.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized as an inflammatory demyelinating disease. Given the need for improvements in MS treatment, many studies are mainly conducted through preclinical models such as experimental allergic encephalomyelitis (EAE). This study analyzes the relationships between histopathological and clinical score findings at EAE. Twenty-three female Rattus norvegicus Lewis rats from 6 to 8 weeks were induced to EAE. Nineteen rats underwent EAE induction distributed in six groups to establish the evolution of clinical signs, and four animals were in the control group. Bordetella pertussis toxin (PTX) doses were 200, 250, 300, 350 and 400 ng. The clinical scores of the animals were analyzed daily, from seven to 24 days after induction. The brains and spinal cords were collected for histopathological analyses. The results demonstrated that the dose of 250 ng of PTX induced a higher clinical score and reduction in weight. All induced groups demonstrated leukocyte infiltration, activation of microglia and astrocytes, and demyelinated plaques in the brains in histopathology. It was concluded that the dose of 250 ng and 350 ng of PTX were the best choices to trigger the brain and spinal cord demyelination lesions and did not correlate with clinical scores.

Produção do Oligômero B da toxina pertussis para uso como adjuvante: etapas iniciais e perspectivas / Production of Oligomer B of pertussis toxin for use as an adjuvant: initial steps and perspectives

Butantan Institute produces a whole cell pertussis vaccine through the fermentation, purification and inactivation processes of Bordetella pertussis cells. B. pertussis is a gram- negative cocobacillus that produces several virulence factors, including adhesins and toxins. Pertussis toxin (PT) is a major virulence factor of B. pertussis and it is considered a key component of the protective immune response against B. pertussis. It has been suggested that the protective efficacy of the whole-cell vaccine may in part be related to residual active PT, and the B oligomer of PT has already been demonstrated to act as adjuvant. The present work aimed to produce the B oligomer and evaluate it as an adjuvant for vaccines produced at Butantan Institute. To obtain oligomer B, the bacterium B. pertussis was grown in a solid medium Bordet & Gengou followed by cultivation in liquid medium to be inoculated in the horizontal Wave bioreactor. The production cultures were performed with 100 mL of the above inoculum in a total of 1 L fermentation, in the Wave horizontal bioreactor for at least 20 hours with controlled temperature and agitation. During the upstream steps, we performed the in-process control, evaluating the following parameters: pH, dissolved oxygen, opacity, optical density at 590 nm (OD590nm), microscopy (Gram staining) and presence of agglutinogens. After cultivation, in the downstream process, centrifugation was performed followed by sterile filtration of the supernatant. To check the production of PT toxin, SDS- PAGE electrophoresis and Western Blotting analysis were performed using a NIBSC reference PT, JNIH-5 (10 mg/mL) and the primary antibody NIBSC Anti-Pertussis PT JNIH- 12. For purification of the pertussis toxin, we performed ion exchange chromatography, using HiTrap CM Fast Flow column. The sample was applied to the column in 50 mM sodium phosphate buffer containing 2 M urea (equilibration buffer), pH 6.0. Then, PT was eluted from the column with Buffer A (pH 7.4) and after elution of PT, FHA was eluted in a gradient of 0-50% buffer A and B (50 mM sodium phosphate pH 7.4 containing Urea 2 M and 1 M NaCl) in a flow of 3 mL/min. After the samples were eluted, the column was washed with 100% buffer B (5 column volumes). Growth in the Wave horizontal bioreactor resulted in cultures with an opacity of 50 UOp/mL, O.D (590nm) of 2.47 and a change in pH between 6.5 and 7.8 during the time of cultivation. The agglutinogen test showed the presence of Agg1, Agg2 and Agg3. Microscopic analysis revealed characteristic gram-negative cells of B. pertussis. The analysis by SDS-PAGE and Western blotting showed bands compatible with those of the positive control, corresponding to the PT subunits. In chromatography, the proteins were eluted separately from the same column and the SDS-PAGE and Western blotting showed separate bands of PT and FHA. As a perspective, after the purification of oligomer B, we will evaluate its potential as a vaccine adjuvant.

O Instituto Butantan produz uma vacina contra pertussis de células inteiras através dos processos de fermentação, purificação e inativação das células de Bordetella pertussis. B. pertussis é um cocobacilo gram-negativo que produz vários fatores de virulência, incluindo adesinas e toxinas. A toxina pertussis (PT) é um fator de virulência importante de B. pertussis e é considerada um componente-chave da resposta imune protetora contra B. pertussis. Foi sugerido que a eficácia protetora da vacina de células inteiras pode, em parte, estar relacionada à PT ativa residual, e já foi demonstrado que o oligômero B da PT atua como adjuvante. O presente trabalho teve como objetivo produzir o oligômero B purificado a partir de cultivos de B. pertussis a fim de avaliá-lo como adjuvante de vacinas. Para obtenção do oligômero B, a bactéria B. pertussis foi cultivada em meio sólido Bordet & Gengou seguida por cultivos em meio de cultura líquido e por produção em biorreator horizontal Wave. O cultivo de produção em Wave foi realizado num volume total de 1 L de fermentação por 20 horas, com temperatura e agitação controladas. Durante as etapas de upstream, realizamos os controles em processo, avaliando os seguintes parâmetros: pH, oxigênio dissolvido, opacidade, densidade óptica a 590 nm (D.O 590nm), microscopia (coloração de Gram) e presença de aglutinógenos. Após o cultivo, as células foram separadas do sobrenadante através de centrifugação. O sobrenadante foi filtrado estéril em membranas de 0,22 um. Para verificar a presença da toxina PT, foram realizadas eletroforese por SDS-PAGE e análise Western Blotting usando uma PT de referência NIBSC, JNIH-5 (10 mg/mL) e o anticorpo primário NIBSC Anti-Pertussis PT JNIH-12. Para a purificação da toxina pertussis foi usada cromatografia de troca iônica. A amostra foi aplicada à coluna HiTrap CM Fast Flow em tampão 50 mM fosfato de sódio contendo ureia 2 M (tampão de equilíbrio), pH 6,0. Em seguida, a PT foi eluída da coluna com Tampão A (pH 7,4) e após a eluição da PT, a FHA foi eluída em gradiente de 0-50% tampão A e B (50 mM fosfato de sódio pH 7,4, contendo Ureia 2 M e NaCl 1 M), num fluxo de 3 mL/min. Após a eluição das amostras, a coluna foi lavada com 100% tampão B (5 volumes de coluna).O crescimento no biorreator horizontal Wave resultou em cultivos com opacidade de 50 UOp/mL, D.O (590nm) de 2,47 e alteração no pH entre 6,5 e 7,8 durante o tempo de cultivo. O teste de aglutinógenos mostrou a presença de Agg1, Agg2 e Agg3. A análise microscópica revelou células gram-negativas características de B. pertussis. A análise por SDS-PAGE e Western blotting mostrou bandas compatíveis com as do controle positivo, correspondentes às subunidades de PT. Na cromatografia, as proteínas foram eluídas separadamente da mesma coluna, e o SDS–PAGE e o Western blotting mostraram as bandas separadas de PT e FHA. Como perspectiva, após a purificação do oligômero B, avaliaremos seu potencial como adjuvante de vacinas.

Seroprevalencia contra Bordetella pertussis en embarazadas vacunadas y no vacunadas y neonatos en un hospital universitario de la provincia de Buenos Aires / Seroprevalence of Bordetella pertussis among vaccinated and unvaccinated pregnant women and newborn infants in a university hospital of Buenos Aires

Introducción. La tos convulsa es una enfermedad altamente contagiosa causada por Bordetella pertussis. Tiene una alta tasa de morbilidad y mortalidad, especialmente, en los lactantes menores de seis meses de edad. En la Argentina, la incidencia y la mortalidad se han encontrado en aumento en las últimas 3 décadas. Objetivo. Determinar anticuerpos contra Bordetella pertussis en las mujeres embarazadas en el tercer trimestre de la gestación y en el recién nacido, medidos en la sangre del cordón. Métodos. Se disenó un estudio observacional, transversal. El estudio se inició en 2011 cuando la vacunación contra pertussis en la embarazada no estaba incluida en el Calendario Nacional de Vacunación y era opcional. Los anticuerpos se midieron en las madres en el tercer trimestre del embarazo y en la sangre del cordón umbilical al nacer. Las determinaciones de anticuerpos se realizaron con el kit de ELISA humano para IgG toxina pertussis ABCAMR. Se utilizó la prueba de chi² para comparar la prevalencia. Resultados. Se incluyó a 111 madres y a sus bebés, 35 hijos de madres no vacunadas (antes de la implementación de la vacuna en embarazadas) y 76 hijos de madres vacunadas. Los bebés de madres vacunadas presentaron anticuerpos IgG positivos en el 92% (70/76), mientras que los bebés de madres no vacunadas fueron negativos para anticuerpos IgG en el 100% (35/35) con una p < 0,001. Conclusión. En la población de vacunadas del estudio, se observó que sus hijos presentaron anticuerpos IgG positivos en el 92%. Este estudio apoya la necesidad de la inmunización materna contra Bordetella pertussis para proteger al recién nacido.

Introduction. Pertussis is a highly contagious disease caused by Bordetella pertussis. It poses a high morbidity and mortality rate, especially among infants younger than 6 months old. In Argentina, pertussis incidence and mortality have increased over the past three decades. Objective. To establish Bordetella pertussis antibody titers among pregnant women in their third trimester and among newborn infants, as measured in cord blood. Methods. This was an observational, crosssectional study. The study started in 2011; at that time, pertussis vaccination was not mandatory for pregnant women as per the national immunization schedule, only optional. Maternal antibodies were measured in the last trimester of pregnancy for women and in cord blood for newborn infants. Antibody titers were determined using Abcam's anti-Bordetella pertussis toxin (PT) IgG in vitro ELISA kit. The X2 test was used to compare prevalence rates. Results. The study included 111 mother-newborn infant dyads; 35 infants from unvaccinated mothers (before the introduction of the vaccine) and 76 from vaccinated mothers. Positive IgG antibodies were found in 92% (70/76) of infants born from vaccinated mothers whereas 100% (35/35) of infants born from unvaccinated mothers had negative results for antibodies; p < 0.001. Conclusion. In the vaccinated population of this study, 92% of infants had positive IgG antibodies. This study supports the need for maternal immunization against Bordetella pertussis to provide protection to newborn infants.

Seroprevalence of Bordetella pertussis among vaccinated and unvaccinated pregnant women and newborn infants in a university hospital of Buenos Aires. / Seroprevalencia contra Bordetella pertussis en embarazadas vacunadas y no vacunadas y neonatos en un hospital universitario de la provincia de Buenos Aires.

INTRODUCTION: Pertussis is a highly contagious disease caused by Bordetella pertussis. It poses a high morbidity and mortality rate, especially among infants younger than 6 months old. In Argentina, pertussis incidence and mortality have increased over the past three decades. OBJETIVE: To establish Bordetella pertussisantibody titers among pregnant women in their third trimester and among newborn infants, as measured in cord blood. METHODS: This was an observational, cross-sectional study. The study started in 2011; at that time, pertussis vaccination was not mandatory for pregnant women as per the national immunization schedule, only optional. Maternal antibodies were measured in the last trimester of pregnancy for women and in cord blood for newborn infants. Antibody titers were determined using Abcam's anti-Bordetella pertussis toxin (PT) IgG in vitro ELISA kit. The χ² test was used to compare prevalence rates. RESULTS: The study included 111 mother-newborn infant dyads; 35 infants from unvaccinated mothers (before the introduction of the vaccine) and 76 from vaccinated mothers. Positive IgG antibodies were found in 92% (70/76) of infants born from vaccinated mothers whereas 100% (35/35) of infants born from unvaccinated mothers had negative results for antibodies; p < 0.001. CONCLUSION: In the vaccinated population of this study, 92% of infants had positive IgG antibodies. This study supports the need for maternal immunization against Bordetella pertussis to provide protection to newborn infants.

INTRODUCCIÓN: La tos convulsa es una enfermedad altamente contagiosa causada por Bordetella pertussis. Tiene una alta tasa de morbilidad y mortalidad, especialmente, en los lactantes menores de seis meses de edad. En la Argentina, la incidencia y la mortalidad se han encontrado en aumento en las últimas 3 décadas. OBJETIVO: Determinar anticuerpos contra Bordetella pertussis en las mujeres embarazadas en el tercer trimestre de la gestación y en el recién nacido, medidos en la sangre del cordón. MÉTODOS: Se diseñó un estudio observacional, transversal. El estudio se inició en 2011 cuando la vacunación contra pertussis en la embarazada no estaba incluida en el Calendario Nacional de Vacunación y era opcional. Los anticuerpos se midieron en las madres en el tercer trimestre del embarazo y en la sangre del cordón umbilical al nacer. Las determinaciones de anticuerpos se realizaron con el kit de ELISA humano para IgG toxina pertussis ABCAM®. Se utilizó la prueba de chi2 para comparar la prevalencia. RESULTADOS: Se incluyó a 111 madres y a sus bebés, 35 hijos de madres no vacunadas (antes de la implementación de la vacuna en embarazadas) y 76 hijos de madres vacunadas. Los bebés de madres vacunadas presentaron anticuerpos IgG positivos en el 92% (70/76), mientras que los bebés de madres no vacunadas fueron negativos para anticuerpos IgG en el 100% (35/35) con una p < 0,001. CONCLUSIÓN: En la población de vacunadas del estudio, se observó que sus hijos presentaron anticuerpos IgG positivos en el 92%. Este estudio apoya la necesidad de la inmunización materna contra Bordetella pertussis para proteger al recién nacido.

Bordetella pertussis modulates human macrophage defense gene expression.

Bordetella pertussis, the etiological agent of whooping cough, still causes outbreaks. We recently found evidence that B. pertussis can survive and even replicate inside human macrophages, indicating that this host cell might serve as a niche for persistence. In this work, we examined the interaction of B. pertussis with a human monocyte cell line (THP-1) that differentiates into macrophages in culture in order to investigate the host cell response to the infection and the mechanisms that promote that intracellular survival. To that end, we investigated the expression profile of a selected number of genes involved in cellular bactericidal activity and the inflammatory response during the early and late phases of infection. The bactericidal and inflammatory response of infected macrophages was progressively downregulated, while the number of THP-1 cells heavily loaded with live bacteria increased over time postinfection. Two of the main toxins of B. pertussis, pertussis toxin (Ptx) and adenylate cyclase (CyaA), were found to be involved in manipulating the host cell response. Therefore, failure to express either toxin proved detrimental to the development of intracellular infections by those bacteria. Taken together, these results support the relevance of host defense gene manipulation to the outcome of the interaction between B. pertussis and macrophages.

Evaluation of a new syringe presentation of reduced-antigen content diphtheria, tetanus, and acellular pertussis vaccine in healthy adolescents--A single blind randomized trial.

Reduced-antigen-content diphtheria-tetanus-acellular pertussis (dTpa) vaccine, Boostrix™, is indicated for booster vaccination of children, adolescents and adults. The original prefilled disposable dTpa syringe presentation was recently replaced by another prefilled-syringe presentation with latex-free tip-caps and plunger-stoppers. 671 healthy adolescents aged 10-15 years who had previously received 5 or 6 previous DT(P)/dT(pa) vaccine doses, were randomized (1:1) to receive dTpa booster, injected using the new (dTpa-new) or previous syringe (dTpa-previous) presentations. Immunogenicity was assessed before and 1-month post-booster vaccination; safety/reactogenicity were assessed during 31-days post-vaccination. Non-inferiority of dTpa-new versus dTpa-previous was demonstrated for all antigens (ULs 95% CIs for GMC ratios ranged between 1.03-1.13). 1-month post-booster, immune responses were in similar ranges for all antigens with both syringe presentations. dTpa delivered using either syringe presentation was well-tolerated. These clinical results complement the technical data and support the use of the new syringe presentation to deliver the dTpa vaccine.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.

BACKGROUND: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10µM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.