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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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In vivo LeTx neutralization.Rats (n≥6 for each group) were i.m. administered with the indicated doses of (A) cAb 29 or (B) mAb 29, followed by i.v. challenge with LeTx (20 µg PA and 10 µg LF), 17 hours later. Animal survival was monitored for the next 24 hours.
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pone-0006351-g003: In vivo LeTx neutralization.Rats (n≥6 for each group) were i.m. administered with the indicated doses of (A) cAb 29 or (B) mAb 29, followed by i.v. challenge with LeTx (20 µg PA and 10 µg LF), 17 hours later. Animal survival was monitored for the next 24 hours.

Mentions: To further explore the PA-neutralizing activity of cAb 29, its in vivo protection capacity against a LeTx challenge was tested. In this assay, antibodies were administered (i.m.) at different doses to rats, followed by an i.v. challenge with a lethal dose of LeTx 17 hrs later. All untreated control animals succumbed to this challenge, with mean time to death (MTTD) of 110 min (Fig. 3A). Treatment of animals with up to 100 µg/kg of cAb 29 did not improve survival rate, nor significantly prolong the MTTD (123 min). However, by increasing the treatment doses to 150 and 200 µg/kg, survival rates of 30 and 55% were attained (Fig. 3A). Full protection of challenged rats was achieved by a passive transfer of 250 µg/kg cAb 29. According to these results, a dose of 170 µg/kg cAb 29 was needed in order to provide 50% protection (PD50). The PA-neutralizing activity of mAb 29 was also evaluated under the same conditions as its chimeric counterpart, demonstrating a similar dose response (PD50 of 160 µg/kg; Fig. 3B). These results are in line with the in vitro assay results, suggesting that the chimerization process had no deleterious effect on the functional activity of the antibody.


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)

In vivo LeTx neutralization.Rats (n≥6 for each group) were i.m. administered with the indicated doses of (A) cAb 29 or (B) mAb 29, followed by i.v. challenge with LeTx (20 µg PA and 10 µg LF), 17 hours later. Animal survival was monitored for the next 24 hours.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006351-g003: In vivo LeTx neutralization.Rats (n≥6 for each group) were i.m. administered with the indicated doses of (A) cAb 29 or (B) mAb 29, followed by i.v. challenge with LeTx (20 µg PA and 10 µg LF), 17 hours later. Animal survival was monitored for the next 24 hours.
Mentions: To further explore the PA-neutralizing activity of cAb 29, its in vivo protection capacity against a LeTx challenge was tested. In this assay, antibodies were administered (i.m.) at different doses to rats, followed by an i.v. challenge with a lethal dose of LeTx 17 hrs later. All untreated control animals succumbed to this challenge, with mean time to death (MTTD) of 110 min (Fig. 3A). Treatment of animals with up to 100 µg/kg of cAb 29 did not improve survival rate, nor significantly prolong the MTTD (123 min). However, by increasing the treatment doses to 150 and 200 µg/kg, survival rates of 30 and 55% were attained (Fig. 3A). Full protection of challenged rats was achieved by a passive transfer of 250 µg/kg cAb 29. According to these results, a dose of 170 µg/kg cAb 29 was needed in order to provide 50% protection (PD50). The PA-neutralizing activity of mAb 29 was also evaluated under the same conditions as its chimeric counterpart, demonstrating a similar dose response (PD50 of 160 µg/kg; Fig. 3B). These results are in line with the in vitro assay results, suggesting that the chimerization process had no deleterious effect on the functional activity of the antibody.

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