A novel high-throughput assay to quantify the vaccine-induced inhibition of Bordetella pertussis adhesion to airway epithelia.

BACKGROUND: Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. RESULTS: We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. CONCLUSIONS: We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.

Vacunación frente a la tos ferina en el adolescente y el adulto / Whooping cough vaccination in adolescents and adults

La tos ferina continúa siendo un problema importante desalud pública, a pesar de que se dispone de vacunaseficaces desde hace más de 50 años. En los últimos añosse ha observado un resurgimiento de esta infección enalgunos países, incluso en los que disponen de coberturasvacunales elevadas, que se asocia con un aumento decasos en adolescentes y adultos. Este incremento seexplica por la disminución de la inmunidad vacunal ynatural con el paso del tiempo, y por el descenso de laincidencia de la enfermedad debido a las campañas devacunación, lo que ha producido una disminución delefecto booster inducido por la infección natural. Lasvacunas antipertusis confieren una inmunidad de cortaduración; a los 4 años de la última dosis, la eficacia vacunales del 84% y disminuye hasta el 46% a los 7 años. Portanto, si se considera que en la mayoría de los países laúltima dosis de vacuna DTP se administra a los 4-6 años de edad, es previsible que, si no hay una exposición natural a Bordetella pertussis, sólo la mitad de los inmunizados estarán protegidos al llegar a la adolescencia, y el número de personas susceptibles aumentará con la edad.La reciente comercialización de vacunas acelulares concomponente antigénico reducido, para uso enadolescentes y adultos (dTpa), va a permitir un mejorcontrol de esta infección. La vacunación universal de losadolescentes es ya una realidad en algunos países. Lasestrategias de vacunación del adulto son más difíciles deimplementar, aunque hay un amplio consenso en losgrupos de riesgo prioritarios

Whooping cough continues to be a major public healthproblem, even though effective vaccines have beenavailable for more than 50 years. In the last few years,there has been a re-emergence of this infection in somecountries, even in those with high vaccination coverage,associated with an increase in cases in adolescents andadults. This increase is explained by the decrease innatural and vaccine immunity with the passage of timeand by the reduction in the incidence of this disease due tovaccination campaigns, which have reduced the boostereffect induced by natural infection. Pertussis vaccinesconfer short-term immunity; 4 years after the last dose,vaccine efficacy is 84%, decreasing to 46% after 7 years.Therefore, since the last dose of the diphtheria-tetanuspertussis(DTP) vaccine is administered at the age of 4-6years in most countries, if there is no natural exposure to Bordetella pertussis, only half of the vaccinated individuals will be protected on reaching adolescence, and the number of susceptible individuals will increase with age.The recent entry on to the market of acellular vaccineswith a reduced antigenic component for use in adolescentsand adults (DTaP), will allow better control of thisinfection. Universal vaccination of adolescents is already being carried out in many countries. Vaccination strategies in adults are more difficult to implement, although there is wide consensus in priority risk groups

Detection of residual pertussis toxin in vaccines using a modified ribosylation assay.

Pertussis toxin (PTx) in its detoxified form is an important component of both whole cell and acellular pertussis vaccines (ACVs). For safety reasons, it is imperative to ensure that the quantity of residual PTx in vaccines does not exceed permissible levels. The majority of the toxic effects of PTx have been attributed to the consequences of PTx-catalyzed ribosylation of the alpha-subunits of signal-transducing guanine-nucleotide-binding proteins. In this report PTx ribosylation activity was determined by an improved enzymatic-high performance liquid chromatography coupled assay using a fluorescein labeled Galpha(i3)C20 peptide. The effect of aluminum salts and other vaccine components on the assay system were also studied. The enzymatic assay system was shown to be a convenient, sensitive method and correlate well with the toxicity observed in vivo by the histamine sensitization assay. This method forms the basis of a new assay which could replace the unsatisfactory animal test currently used in pertussis vaccines control.

A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.

Key points for the development of mouse immunogenicity test as potency assay for acellular pertussis vaccines.

According to WHO and the European Pharmacopoeia, the current potency test for acellular pertussis vaccines is a mouse immunogenicity assay assessing consistency of production from batch to batch. The assay compares the batch under control with a reference vaccine of documented clinical efficacy. This study describes and illustrates critical aspects of the assay, based on our experience on a tricomponent vaccine: validation of immunoassay to quantify mouse antibody response, choice of vaccine immunising doses in the three-doses model, treatment of non-responder mice for calculations, establishment of assay validity criteria.