50% lysis of the vaccine strain (B:4:P1.19,15). The results against heterologous strains were poor. Taking into account these and other results we evaluated the behavior of the WBA using lower concentrations of the bacterial suspension used as inoculum. Blood from two healthy adults was screened for bactericidal activity using the WBA, against B:4:P1.19,15(Cu 385/83) and B:15:P1.7,16 (MC58) strains. In addition to the recommended concentration (106 or 107 CFU/mL), two lower concentrations were used (105 and 104 CFU/mL). The first donor showed a lytic activity of 40% against the homologous strain, at a concentration of 107 CFU/mL. At 105 and 104 CFU/mL concentrations, lysis was around 80%. The behavior against the heterologous strain showed 25% of lysis at a concentration of 107 CFU/mL and around 60% at lower concentrations. The second donor did not show lytic activity against the homologous strain at a concentration of 107 CFU/mL, while it showed 55% and 65% lysis at 105 and 104 CFU/mL concentrations, respectively. The results against the heterologous strain were 40% of lysis for the highest concentration and 70% and 80% for concentrations of 105 and 104 CFU/mL respectively.]]>
Resultados preliminares de la evaluación de diferentResultados diferentes concentraciones de la suspensión bacteriana empleada como inóculo en el Ensayo bactericida de Sangre Total.
Preliminary evaluation of different concentrations of bacterial suspensions used in the Whole Blood Bactericidal Assay.
María Amalia Camaraza1, Natasha Anwar2, Teresita Leiva1, Aida G. Arnet1, Franklin Sotolongo1,
Catherine Ison2.
1 Instituto Finlay. Centro de Investigación-Producción de Vacunas y Sueros. La Habana, Cuba. E-mail: camaraza@finlay.edu.cu. ]]> 2 Saint Mary´s Hospital, Imperial College Medical School. Londres, Reino Unido.
RESUMEN
El Ensayo Bactericida del Suero (EBS), está considerado como la “prueba de oro” para evaluar la eficacia serológica de vacunas antimeningocócicas, teniendo en cuenta que la presencia de anticuerpos bactericidas en el suero se relaciona con la protección frente a esta enfermedad. Los niveles de anticuerpos anticapsulares obtenidos frente a los serogrupos A y C de Neisseria meningitidis han confirmado la utilidad de esta técnica. Sin embargo, se señala una pobre correlación entre los resultados de laboratorio y la protección clínica demostrada después de la aplicación de vacunas compuestas por vesículas de membrana externa del serogrupo B. Ison y colaboradores desarrollaron el Ensayo de Sangre Total (EST), que evalúa la capacidad bactericida de la sangre. Los resultados obtenidos indican que es un marcador de inmunidad más sensible que el EBS para el serogrupo B. En un estudio de evaluación de la vacuna antimeningocócica cubana VA-MENGOC-BC®, realizado en infantes y empleando el EST, se obtuvo un 50% de respondedores a la cepa homóloga (B:4:P1.19,15). La respuesta contra cepas heterólogas resultó baja. Teniendo en cuenta estos resultados y los de otros estudios realizados por los autores, se evaluó el comportamiento de la prueba utilizando concentraciones menores de la suspensión bacteriana empleada como inóculo. Para este propósito se estudió la actividad bactericida de la sangre de dos adultos sanos, mediante el EST frente a las cepas B:4:P1.19,15 (Cu 385/83) y B:15:P1.7,16 (MC58). Se evaluaron, además de la concentración recomendada originalmente (106ó107 UFC/mL), dos inferiores (105 y 104 UFC/mL). El primer donante mostró una actividad lítica del 40% frente a la cepa homóloga, a una concentración de inóculo de 107 UFC/mL. Para las concentraciones de 105 y 104 UFC/mL la lisis fue de alrededor del 80%. El comportamiento frente a la cepa heteróloga fue de un 25% de lisis a la mayor concentración del inóculo y aproximadamente de un 60% para las concentraciones inferiores. El segundo donante no mostró actividad lítica alguna frente a un inóculo de 107 UFC/mL de la cepa homóloga, mientras que empleando inóculos de 105 y 104 UFC/mL se obtuvieron respuestas de alrededor del 55% y del 65% respectivamente. El comportamiento frente a la cepa heteróloga fue de un 40% de lisis a la mayor concentración del inóculo y de un 70% y 80% a las concentraciones 105 y 104 UFC/mL respectivamente.
Palabras claves: Ensayo Bactericida del Suero, Ensayo de Sangre Total, Neisseria meningitidis.
ABSTRACT
The Serum Bactericidal Assay (SBA) has been considered the “golden standard” to evaluate the serological efficacy of meningococcal vaccines, taking into account that the presence of serum bactericidal antibodies is related with protection. The levels of A and C capsular polysaccharide antibodies against these serogroups has confirmed the reliability of this assay. The SBA has also been used to asses the efficacy of serogroup B vaccines, but in some studies, the correlation between serological efficacy and protection has been poor. Ison et al developed the Whole Blood Assay (WBA) that measures the complete bactericidal activity in blood. The results obtained indicate that this model is a more sensitive marker of immunity than SBA for serogroup B. The results from the evaluation of the Cuban meningococcal vaccine (VA-MENGOC-BC®) in infants using WBA showed that after the immunisation around 50% of infants exhibited >50% lysis of the vaccine strain (B:4:P1.19,15). The results against heterologous strains were poor. Taking into account these and other results we evaluated the behavior of the WBA using lower concentrations of the bacterial suspension used as inoculum. Blood from two healthy adults was screened for bactericidal activity using the WBA, against B:4:P1.19,15(Cu 385/83) and B:15:P1.7,16 (MC58) strains. In addition to the recommended concentration (106 or 107 CFU/mL), two lower concentrations were used (105 and 104 CFU/mL). The first donor showed a lytic activity of 40% against the homologous strain, at a concentration of 107 CFU/mL. At 105 and 104 CFU/mL concentrations, lysis was around 80%. The behavior against the heterologous strain showed 25% of lysis at a concentration of 107 CFU/mL and around 60% at lower concentrations. The second donor did not show lytic activity against the homologous strain at a concentration of 107 CFU/mL, while it showed 55% and 65% lysis at 105 and 104 CFU/mL concentrations, respectively. The results against the heterologous strain were 40% of lysis for the highest concentration and 70% and 80% for concentrations of 105 and 104 CFU/mL respectively.
Key words: Serum Bactericidal Assay, Whole Blood Assay, Neisseria meningitidis
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