Murine monoclonal antibodies neutralizing the cytotoxic activity of diphtheria toxin.

In this study, murine monoclonal antibodies that specifically bound to the A and B subunits of diphtheria toxin (DT) were produced by conventional hybridoma technology using the spleens of BALB/c mice immunized with diphtheria DTP vaccine and CRM197. Monoclonal antibodies specific to the A subunit, i.e. clone AC5, as well as those specific to the B subunit, i.e. clone BB7, could neutralize the DT-mediated cytotoxicity to Vero cells in microcultures. The DT neutralizing mechanisms have yet to be determined. The MAbBB7 is hypothesized to either interfere with the DT receptor binding or with the pore forming function of the T domain of the B subunit. The MAbAC5 could neutralize the DT mediated cytotoxicity when mixed with the DT before adding to the Vero cell culture thus suggesting that the antibody interfered with the translocation of the A subunit. The A subunit-antibody complex might be too large to pass through the membrane channel formed by the T domain and thus prevent the accessibility of the A subunit to the cytosolic target. It is also possible that the MAb AC5 blocked the enzymatic active site of the enzyme catalytic subunit. While further experiments are needed to localize the epitopes of the two MAbs on the holo-DT in order to reveal the DT neutralizing mechanisms, both MAbs in their humanized forms have a high potential as human therapeutic antibodies for diphtheria.

Monoclonal antibody that neutralizes pertussis toxin activities.

Pertussis or whooping cough is a disease with high mortality among infants and small children. The disease is caused by infection of the respiratory tract by a gram negative bacterium, Bordetella pertussis. The superficial colonized bacteria produce a myriad of toxins which enter the circulation causing various pathophysiologicalal changes in the host. Although antimicrobial therapy reduces the number of the coughed out bacteria and also the infectious time of the infected host, but it is not effective in amelioration of the clinical manifestations as the pertussis morbidity is due principally to the pertussis toxin (PT). Antibody based-therapy is frequently practiced in conjunction with other supportive measure to resuscitate the patient. Nevertheless, human derived antiserum against PT is of the limited supply and the ethical concern. Thus in this study a hybridoma clone, i.e. clone PT6-2G6, secreting monoclonal antibody (MAb) specific to the S1 subunit, the active enzyme of the PT that intracellularly ADP-ribosylates the host Gi-protein, was produced. The MAbPT6-2G6 inhibited the in vitro hemagglutination of chicken erythrocytes which is the activity of the B oligomer of PT; thus we hypothesize that the MAb bound to its epitope on the S1 subunit and stereologically hinders the binding sites of the B subunits. The MAb also inhibited ex vivo Chinese hamster ovarian cell clustering and neutralized the in vivo leucocytosis- promotion in mice which are usually mediated by intracellular S1 subunit. The large molecular nature of the intact MAb and its molecular hydrophilicity led us to speculate that the observed PT neutralizing activities of the MAb were due to interfering with the cellular entry of the S1 rather than the intracellular enzyme neutralizing activity per se. While further experiments are needed to pinpoint the MAb neutralizing activity and to identify the amino acid sequence and location of the MAbPT6-2G6 epitope, our findings indicate that this murine MAb, in its humanized-version, should have high therapeutic potential for pertussis.