Comparison of Three Anthrax Toxin Neutralization Assays

Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays—J774A.1 cell-, RAW 264.7 cell-, and CHO cell-based assays—yield comparable estimates of neutralization activities for sera obtained after vaccination with anthrax vaccines composed of recombinant protective antigen (rPA). In order to compare the assays, sera were assayed alongside a common reference serum sample and the neutralization titers were expressed relative to the titer for the reference sample in each assay. Analysis of sera from rabbits immunized with multiple doses of the rPA vaccine showed that for later bleeds, the quantitative agreement between the assays was good; however, for early bleeds, some heterogeneity in relative neutralization estimates was observed. Analysis of serum samples from rabbits, nonhuman primates, and humans immunized with the rPA vaccine showed that the relative neutralization estimates obtained in the different assays agreed to various extents, depending on the species of origin of the sera examined. We identified differences in the magnitudes of the Fc receptor-mediated neutralization associated with the J774A.1 cell- and RAW 264.7 cell-based assays, which may account for some of the species dependence of the assays. The differences in the relative neutralization estimates among the assays were relatively small and were always less than 2.5-fold. However, because toxin neutralization assays will likely be used to establish the efficacies of new anthrax vaccines, our findings should be considered when assay outputs are interpreted.Inhalation anthrax is one of the most serious of all bioterror threats because of the fatal nature of the disease and the stability and ease of dispersion of Bacillus anthracis spores. Therefore, significant efforts are under way to develop new vaccines for the prevention of anthrax. Many of the new anthrax vaccines being developed specifically target anthrax toxin, which is believed to play a critical role in disease progression and the lethal nature of the disease (1, 4, 14, 18, 26).Anthrax toxin is a tripartite toxin, composed of a binary combination of three proteins: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA, which by itself lacks toxic activity, combines with LF, a zinc-dependent metalloprotease, to form lethal toxin (LT) and with EF, a calmodulin- and calcium-dependent adenylate cyclase, to form edema toxin (ET). PA binds to cell receptors and mediates the translocation of LF and EF into the cytosol, where LF inactivates mitogen-activated protein kinase kinases, and EF elevates the levels of cellular cyclic AMP (cAMP) (3, 5, 12).Given the role played by the toxin in the pathology of anthrax, neutralization of the toxin would be expected to prevent or ameliorate anthrax disease. Indeed, numerous animal and in vitro studies have shown that antibodies to toxin components and, in particular, PA confer protection (8, 11, 15, 23, 34). For this reason, many of the new anthrax vaccines under development are based on PA.Because of the low incidence of anthrax disease in humans, the conduct of human efficacy trials of new anthrax vaccines is not feasible. Challenge studies with humans would be unethical because of the rapid and fatal progression of the disease. Therefore, the approval of new anthrax vaccines by the Food and Drug Administration will be based on the Animal Rule (6). Under this regulation, protection data from relevant animal species may be used to support vaccine efficacy in humans. Studies of the pathology of anthrax and the immunogenicity of vaccines in animals have led to the conclusion that both the rabbit and the nonhuman primate (NHP) could serve as suitable animal models of human anthrax and the response to vaccination (7, 22, 30, 35). Anti-PA antibody levels in immune sera have been shown to correlate with protection in animal models (16, 33) and thus will likely be used to bridge animal protection data to efficacy in humans. Thus, antibody levels will be important in establishing vaccine efficacy, and the assays used to measure these levels are critical to this process.Two types of serological assays, an anti-PA IgG enzyme-linked immunosorbent assay (ELISA) and the anthrax toxin neutralizing assay (TNA) have been utilized to determine the antibody levels elicited by anthrax vaccines (17, 21, 24, 25). While the anti-PA ELISA measures the total amount of anti-PA IgG in a serum sample, TNA quantifies the anti-PA antibodies that are capable of neutralizing the toxin. Thus, TNA measures the subset of antibodies that are considered functional. Moreover, TNA is considered to be species independent and has been standardized for use with multiple species (10, 13, 20, 36). A species-neutral attribute is important for an assay that is to be used to bridge animal protection data to efficacy in humans.Three types of TNAs have been developed. Two of the assay formats, the J774A.1 (J774) cell- and the RAW 264.7 (RAW) cell-based TNAs, measure the ability of anti-PA antibodies to neutralize the cytocidal activity of LT. Both J774A.1 and RAW 264.7 cells are murine macrophage-like cell lines. The third assay format is the CHO cell-based TNA, which measures the ability of anti-PA antibodies to neutralize the ET-induced increase in intracellular cAMP levels. Both the specific toxin and the cell line utilized in the CHO cell-based assay differ from those used in the assays of cytocidal activity. The RAW 264.7 cell-based assay has primarily been used in research studies (see, e.g., references 2 and 19). In addition to their use in research studies, the J774A.1 cell- and CHO cell-based assays have been used to assay serum samples from clinical trials to ascertain the immunogenicities of anthrax vaccines and anti-PA therapeutic monoclonal antibody (MAb) levels, respectively (9, 28).The general assumption is that in all the three TNAs, the antibodies elicited by PA-based vaccines recognize the common PA antigen and, therefore, that all three assays should yield the same relative estimate of toxin neutralization activity compared to that of an appropriate reference sample. However, differences in the specific toxin and cell types used in the three assays could result in differences in estimates of neutralizing antibody titers. For example, differences in the specific toxin utilized in the assay (LT versus ET) might affect neutralization estimates if the anti-PA antibodies present in a given serum sample were better able to inhibit the interaction of one of the catalytic components, either LF or EF, with PA than the other. Differences in cell type might also affect estimates of neutralization, since mechanisms of toxin neutralization can differ depending on the cell type. For example, previous studies have shown that cells of myeloid origin, such as J774A.1 and RAW 264.7 cells, express Fc receptors that can contribute to toxin neutralization. Thus, these cells have the potential to display the “classical” toxin neutralization that is seen with other cell types, as well as an Fc receptor-mediated form of toxin neutralization (31, 32). With classical neutralization, antibodies bind to PA and disrupt one or more of the steps involved in toxin action. Fc receptor-mediated neutralization is not well understood but could be due to Fc receptor-bound anti-PA facilitating the endocytosis of immune complexes, leading to their degradation, the sequestration of PA monomers at the cell surface, and/or the enhancement of classical neutralization by increasing the effective concentration of neutralizing antibodies at the cell surface. While Fc receptor-mediated neutralization occurs with both J774A.1 and RAW 264.7 cells, the extent of this type of neutralization differs between cell types (31). Unlike with these macrophage-like cell lines, Fc receptor-mediated neutralization is not expected to occur with CHO cells, an epithelial cell line, since only cells of hematopoietic origin produce Fc receptors.In this study, we sought to determine whether the three TNAs yield comparable estimates of neutralization activity for serum samples. We chose to compare the three TNA formats first by using sera generated in rabbits after the administration of either one, two, or three doses of a recombinant PA (rPA) vaccine in order to determine whether differences in estimates of neutralizing antibody titers might depend on the number of doses of vaccine administered. In addition, we compared the three assay formats using sera from three different species—rabbits, NHPs, and humans—to determine whether the species of origin of the sera might influence neutralization estimates. We conducted the latter comparison since the TNA will be used to bridge rabbit/NHP protection data to efficacy in humans. Our goal was to determine whether the three assays yield similar estimates of neutralization and thus assess if they can serve equally well to measure the toxin-neutralizing antibody titers in pivotal animal protection studies as well as in human clinical trials.