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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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Related in: MedlinePlus

Kinetics of anti-PA and PA-neutralizing antibody development.Average titer values of total anti-PA ELISA antibodies (filled circles) of eight immunized mice and LeTx neutralizing antibody titers (empty circles) of mice 3, 4, 5 and 7, throughout the immunization process. Arrows indicate PA-immunization time points; Data points are mean±STD.
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pone-0006351-g001: Kinetics of anti-PA and PA-neutralizing antibody development.Average titer values of total anti-PA ELISA antibodies (filled circles) of eight immunized mice and LeTx neutralizing antibody titers (empty circles) of mice 3, 4, 5 and 7, throughout the immunization process. Arrows indicate PA-immunization time points; Data points are mean±STD.

Mentions: Mice were immunized with a PA-based vaccine, and titer development of either PA-neutralizing or PA-binding antibodies was carefully monitored. Two weeks after the third PA-administration (week 6), all immunized mice had developed high anti-PA titers of 30,000–60,000 (Fig. 1). Yet, at this time point, no measurable neutralizing antibody titer could be detected, and therefore another boost was given. Indeed, in four out of the eight immunized mice, neutralizing antibody titer developed gradually, reaching a maximum titer value of 30,000 by week 10. Five weeks later, the neutralizing antibody titer remained constant in these four mice (Fig. 1), yet remained negative for the other four immunized mice (data not shown). It was noticeable that, although PA-neutralizing antibodies titer increased several-fold in these mice, the titer of the total anti-PA antibodies remained unchanged. These results suggest that the continuous exposure to PA-based vaccine in these mice may have led to a specific enrichment and maturation of the desired subset of PA-neutralizing antibodies.


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)

Kinetics of anti-PA and PA-neutralizing antibody development.Average titer values of total anti-PA ELISA antibodies (filled circles) of eight immunized mice and LeTx neutralizing antibody titers (empty circles) of mice 3, 4, 5 and 7, throughout the immunization process. Arrows indicate PA-immunization time points; Data points are mean±STD.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006351-g001: Kinetics of anti-PA and PA-neutralizing antibody development.Average titer values of total anti-PA ELISA antibodies (filled circles) of eight immunized mice and LeTx neutralizing antibody titers (empty circles) of mice 3, 4, 5 and 7, throughout the immunization process. Arrows indicate PA-immunization time points; Data points are mean±STD.
Mentions: Mice were immunized with a PA-based vaccine, and titer development of either PA-neutralizing or PA-binding antibodies was carefully monitored. Two weeks after the third PA-administration (week 6), all immunized mice had developed high anti-PA titers of 30,000–60,000 (Fig. 1). Yet, at this time point, no measurable neutralizing antibody titer could be detected, and therefore another boost was given. Indeed, in four out of the eight immunized mice, neutralizing antibody titer developed gradually, reaching a maximum titer value of 30,000 by week 10. Five weeks later, the neutralizing antibody titer remained constant in these four mice (Fig. 1), yet remained negative for the other four immunized mice (data not shown). It was noticeable that, although PA-neutralizing antibodies titer increased several-fold in these mice, the titer of the total anti-PA antibodies remained unchanged. These results suggest that the continuous exposure to PA-based vaccine in these mice may have led to a specific enrichment and maturation of the desired subset of PA-neutralizing antibodies.

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