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Clinical and Vaccine Immunology, October 2009, p. 1405-1412, Vol. 16, No. 10
1071-412X/09/$08.00+0     doi:10.1128/CVI.00194-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays{triangledown}

Anita Verma,1 Miriam M. Ngundi,1 Bruce D. Meade,2 Roberto De Pascalis,1 Karen L. Elkins,1 and Drusilla L. Burns1*

Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892,1 Meade Biologics, Hillsborough, North Carolina 272782

Received 15 May 2009/ Returned for modification 18 June 2009/ Accepted 30 July 2009

Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fc{gamma}) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fc{gamma} receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fc{gamma} receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fc{gamma} receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fc{gamma} receptor blocking monoclonal antibodies, we found that at least three murine Fc{gamma} receptor classes, IIB, III, and IV, can contribute to Fc{gamma} receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fc{gamma} receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fc{gamma} receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output.

* Corresponding author. Mailing address: CBER, FDA, HFM-434, Building 29, Room 130, 8800 Rockville Pike, Bethesda, MD 20892. Phone: (301) 402-3553. Fax: (301) 402-2776. E-mail: drusilla.burns{at}fda.hhs.gov

{triangledown} Published ahead of print on 5 August 2009.

Clinical and Vaccine Immunology, October 2009, p. 1405-1412, Vol. 16, No. 10
1071-412X/09/$08.00+0     doi:10.1128/CVI.00194-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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