RESUMEN
Five synthetic peptides identified as antigenic sites on the S1 subunit of pertussis toxin (PT) were coupled to the diphtheria toxin cross-reactive mutant protein CRM 197 or BSA. All peptide conjugates were immunogenic in animals. Two peptide-CRM-conjugates, corresponding to amino acids 1-17 and 169-186, induced especially high antibody titers against native PT in mice (Balb/c, C57/Black and outbred NMRI) as measured by ELISA. Upon in vivo PT challenge (0.5 microgram toxin) of the NMRI mice both the CRM and BSA conjugates of these two peptides fully protected the mice from PT induced leucocytosis. Some of the protected mice receiving peptide 1-17 conjugate had very low antibody titers against PT but high titers against the peptide as measured by ELISA, showing that PT-ELISA does not always reflect in vivo protection. A booster response against PT was noted upon challenge with PT in mice receiving peptide 1-17 or peptide 169-186 conjugate. Antibodies against peptide 170-186 could not be evaluated by ELISA since conjugates of this peptide (like PT itself) bind to immunoglobulins. They may also caused clustering of CHO cells. Rabbit antiserum to the peptide 1-17-CRM conjugate was highly efficient in inhibiting the ADP-ribosylating activity of PT with bovine transducin as substrate whereas the rabbit antiserum raised against the peptide 169-186-CRM conjugate neutralized the clustering effect of PT on CHO cells. Thus there is no concise correlation between the in vivo protection against PT challenge and the in vitro methods used for measuring antibody levels against PT (neutralization of the enzyme activity, the CHO cell clustering activity and titers in PT-ELISA). The CRM-conjugates of these two peptides constitute the first synthetic pertussis vaccine candidate with the ability to provide a chemically well defined, safe and efficient pertussis vaccine.
Asunto(s)
Proteínas Bacterianas/inmunología , Toxina del Pertussis , Factores de Virulencia de Bordetella/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Bordetella pertussis/genética , Bordetella pertussis/inmunología , Leucocitosis/inmunología , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/genética , Péptidos/inmunología , Vacuna contra la Tos Ferina/inmunología , Vacunas Sintéticas/inmunología , Factores de Virulencia de Bordetella/genética , Tos Ferina/prevención & controlRESUMEN
Two peptides, corresponding to amino acids 1-17 and 169-186 of the amino acid sequence of pertussis toxin (PT) subunit S1, were synthesized and coupled to the diphtheria toxin cross-reactive mutant protein CRM 197 and evaluated for immunogenicity and protective capacity against PT challenge in vivo. The peptide-CRM conjugates induced high antibody titers against native toxin in mice (BALB/c, C57/Black, and outbred NMRI) as measured by ELISA. Upon PT challenge (0.5 microgram of toxin) of the NMRI mice, the CRM conjugates of peptides 1-17 and 169-186 fully protected the mice from PT-induced leukocytosis. Immunization with the corresponding bovine serum albumin conjugates of these two peptides also fully protected mice. Rabbit antiserum to the peptide 1-17-CRM conjugate was highly efficient in inhibiting the ADP-ribosylating activity of PT but did not neutralize the clustering effect of PT on Chinese hamster ovary cells. In contrast, the rabbit antiserum raised against the peptide 169-186-CRM conjugate neutralized the clustering effect of PT on Chinese hamster ovary cells but did not inhibit the enzymatic activity of PT. Peptide 169-186-CRM conjugates mimic the immunoglobulin binding properties of PT and also cause clustering of Chinese hamster ovary cells. The CRM conjugates of these two peptides constitute a synthetic pertussis vaccine candidate with the ability to provide a chemically well-defined, safe, and efficient pertussis vaccine.
Asunto(s)
Inmunización , Péptidos/inmunología , Toxina del Pertussis , Factores de Virulencia de Bordetella/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/análisis , Formación de Anticuerpos , Toxina Diftérica/genética , Ensayo de Inmunoadsorción Enzimática , Sustancias Macromoleculares , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Mutación , NAD/metabolismo , Péptidos/síntesis química , Tos Ferina/prevención & controlRESUMEN
We synthesized six peptides (15-22 amino acids) from the amino acid sequence of the S1 subunit of pertussis toxin. The antigenicity of the polypeptides was investigated by enzyme-linked immunosorbent assays, in which the polypeptides coupled to bovine serum albumin were used as coating antigens. The polypeptides were examined as antigens for reaction with pre- and postvaccination sera from infants receiving either a Bordetella pertussis whole-cell vaccine or a vaccine containing pertussis toxoid. An elevated serum titer was noted upon vaccination with both types of vaccines when bovine serum albumin conjugates of five synthetic peptides were used as antigens. Similarly, mice immunized with whole-cell pertussis vaccine showed an elevated titer against all five peptides that were reactive with human sera. Thus, we identified five peptide sequences of S1 against which, in two species, antibodies are formed upon exposure of these species to whole cells of B. pertussis.