RESUMEN
Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 microg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Toxina Shiga II/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Células CHO , Chlorocebus aethiops , Cricetinae , Femenino , Humanos , Ratones , Datos de Secuencia Molecular , Toxina Shiga II/toxicidad , Células VeroRESUMEN
Pulmonary infection with Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa toxin is one of several proposed virulence factors which may be responsible for chronic P. aeruginosa infections in these patients. With a highly specific, sensitive, and quantitative radioimmunoassay (RIA) and a cell culture assay, the humoral immune responses of CF patients in terms of total antitoxin, antitoxin immunoglobulins A and M, and neutralizing antitoxin were compared with those of P. aeruginosa-infected intensive care unit patients and controls. The P. aeruginosa-infected CF patients were divided into severe and moderate disease groups based on mortality observed over an 8-year period. The intensive care unit patients were divided by the site of infection and the controls were healthy children and uninfected CF patients. Antibodies to toxin were found in the sera of all subjects by radioimmunoassay. Neutralizing antibody was associated with current infection. Elevated titers of antitoxin immunoglobulin A were found only in subjects with pulmonary P. aeruginosa infections. No significant differences in any antibody class were observed between the severe and moderate disease groups. In addition, no differences were observed in the antitoxin immune response of chronically infected CF patients and intensive care unit patients with acute pulmonary infections.