Analysis of the Fc gamma receptor-dependent component of neutralization measured by anthrax toxin neutralization assays

Abstract

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 (Fcgamma) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcgamma 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 Fcgamma receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcgamma receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcgamma receptor blocking monoclonal antibodies, we found that at least three murine Fcgamma receptor classes, IIB, III, and IV, can contribute to Fcgamma receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcgamma 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 Fcgamma 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.

FIG. 1.

Contribution of Fcγ receptor-dependent neutralization in J774A.1 cell-based and RAW 264.7 cell-based TNAs. IgG was purified from the polyclonal antiserum NR-3839; F(ab′)₂ fragments were prepared from this IgG preparation. The indicated concentrations of either IgG or F(ab′)₂ fragments were examined for their ability to neutralize LT in either the J774A.1 cell-based TNA (A) or the RAW 264.7 cell-based TNA (B). Each point corresponds to the mean of the values obtained for three independent sample preparations run on the same plate, with the standard error of the mean indicated by the error bar. For each independent assay, the samples were run on duplicate plates. The figures are representative of four and three independent assays for the J774A.1 and RAW 264.7 cell-based assays, respectively, with each independent assay run on different days.

FIG. 2.

Quantification of Fcγ receptors on J774A.1 and RAW 264.7 cells, determined using flow cytometry. Flow cytometric analysis was performed as described in Materials and Methods. Representative histograms of live CD45⁺ CD11b⁺ CD19⁻ cells analyzed for FcγRI (C) and FcγRII/III/IV (D) are shown, with comparison to the appropriate isotype-matched, fluorochrome-labeled control antibodies for each (FcγRI [A] and FcγRII/III/IV [B]). The arithmetic mean fluorescent intensities ± standard deviations from triplicate samples are also shown (E).

FIG. 3.

Toxin neutralizing activity of rabbit polyclonal serum NR-3839, measured in the presence and absence of anti-FcγRII/III or anti-FcγRIV. TNAs were performed essentially as described in Materials and Methods. The indicated dilutions of NR-3839 were used to neutralize LT in J774A.1 cell-based TNAs in the absence (−) or presence (+) of anti-FcγRII/III (MAb 2.4G2) (A) and anti-FcγRIV (MAb 9E9) (B). Each point corresponds to the mean of the values obtained for two independent sample preparations each run on a separate plate, with the standard error of the mean indicated by the error bar. Each figure is representative of three independent assays, each run on different days.

FIG. 4.

Fcγ receptor dependence of anthrax toxin neutralization, determined using bone marrow-derived macrophages from wild-type (WT) and KO mice. Primary cultures of bone marrow-derived macrophages were prepared from WT, FcγRIIB KO, and FcγRI/III/IV KO strains of BALB/c mice. The macrophages were exposed to LT and various dilutions of polyclonal rabbit serum NR-3839, either in the absence (−) or in the presence (+) of the FcγRII/III blocking MAb 2.4G2. Each point corresponds to the mean of the values obtained for two independent sample preparations each run on a separate plate, with the standard error of the mean indicated by the error bar. Each figure is representative of three independent assays, each run on different days.

FIG. 5.

Dependence of Fcγ receptor-mediated neutralization on the concentration of PA used in the TNA. The indicated dilutions of NR-3839 in the absence (−) or presence (+) of anti-FcγRII/III (MAb 2.4G2) were examined for their ability to neutralize LT in either the J774A.1 cell-based TNA (A and B) or the RAW 264.7 cell-based TNA (C and D), using a PA concentration of either 50 ng/ml (A and C) or 400 ng/ml (B and D). Each point corresponds to the mean of the values obtained for three independent sample preparations run on the same plate, with the standard error of the mean indicated by the error bar. For each independent assay, the samples were run on duplicate plates. Each figure is representative of three independent assays, each run on different days.