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Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

Rosenfeld R, Marcus H, Ben-Arie E, Lachmi BE, Mechaly A, Reuveny S, Gat O, Mazor O, Ordentlich A - PLoS ONE (2009)

Bottom Line: Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores.The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol.The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona, Israel.

ABSTRACT
Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD(50)B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

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Circulatory clearance profiles of cAb 29 and mAb 29.Guinea pigs (n = 3 for each group) were i.m. administered with 5 mg/kg of either cAb 29 (diamonds) or mAb 29 (squares). Blood samples drawn at various time points were assayed for Ab concentration by ELISA. Mean±STD values are presented as percentages of maximum blood levels (Cmax).
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pone-0006351-g005: Circulatory clearance profiles of cAb 29 and mAb 29.Guinea pigs (n = 3 for each group) were i.m. administered with 5 mg/kg of either cAb 29 (diamonds) or mAb 29 (squares). Blood samples drawn at various time points were assayed for Ab concentration by ELISA. Mean±STD values are presented as percentages of maximum blood levels (Cmax).

Mentions: The chimeric antibody cAb 29 and its parental murine antibody, mAb 29, were administered (i.m.) to guinea pigs, and their clearance profiles were determined by measuring the ELISA-titers of blood samples collected at different time points (Fig. 5). It was found that maximal antibody concentrations (Cmax) in the bloodstream were attained (and subsequently defined as 100% concentrations) 14 hours after injection of both antibodies. In the case of cAb 29, this level remained unchanged for the next 5 days, after which the antibody was gradually cleared from the animals' bloodstreams. The calculated mean resident time (MRT) value of cAb 29 was 6.7 days. In the case of the murine antibody, however, maximal levels were retained for only 24 hrs, displaying an MRT value of 3.3 days.


Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

Rosenfeld R, Marcus H, Ben-Arie E, Lachmi BE, Mechaly A, Reuveny S, Gat O, Mazor O, Ordentlich A - PLoS ONE (2009)

Circulatory clearance profiles of cAb 29 and mAb 29.Guinea pigs (n = 3 for each group) were i.m. administered with 5 mg/kg of either cAb 29 (diamonds) or mAb 29 (squares). Blood samples drawn at various time points were assayed for Ab concentration by ELISA. Mean±STD values are presented as percentages of maximum blood levels (Cmax).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2710523&req=5

pone-0006351-g005: Circulatory clearance profiles of cAb 29 and mAb 29.Guinea pigs (n = 3 for each group) were i.m. administered with 5 mg/kg of either cAb 29 (diamonds) or mAb 29 (squares). Blood samples drawn at various time points were assayed for Ab concentration by ELISA. Mean±STD values are presented as percentages of maximum blood levels (Cmax).
Mentions: The chimeric antibody cAb 29 and its parental murine antibody, mAb 29, were administered (i.m.) to guinea pigs, and their clearance profiles were determined by measuring the ELISA-titers of blood samples collected at different time points (Fig. 5). It was found that maximal antibody concentrations (Cmax) in the bloodstream were attained (and subsequently defined as 100% concentrations) 14 hours after injection of both antibodies. In the case of cAb 29, this level remained unchanged for the next 5 days, after which the antibody was gradually cleared from the animals' bloodstreams. The calculated mean resident time (MRT) value of cAb 29 was 6.7 days. In the case of the murine antibody, however, maximal levels were retained for only 24 hrs, displaying an MRT value of 3.3 days.

Bottom Line: Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores.The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol.The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona, Israel.

ABSTRACT
Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD(50)B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

Show MeSH
Related in: MedlinePlus