On the basis of the current interest and performance of the TNA in clinical and nonclinical studies, the assay, in either the J774A.1 cell-, RAW 264.7 cell-, or CHO cell-based format, will continue to be utilized in immunogenicity studies of anthrax vaccines as well as in the evaluation of immunotherapies for anthrax. It is therefore important to determine how the three assays compare in their capacities to estimate the neutralization abilities of various antibodies. Understanding the performance of each assay is critical to choosing the most appropriate assay format and for interpretation of the data.
In our study, the three TNAs yielded various results for the estimates of neutralization for sera (relative to the estimate for the reference serum sample) throughout the course of immunization of rabbits with three doses of rPA. The level of agreement between the assays was good for sera collected after the administration of either two or three doses of the rPA vaccine. After a single immunization, estimates of neutralization differed between the assays by as much as 2-fold. In addition, some heterogeneity in the RP ratios for individual serum samples was observed, especially for the J774/CHO RP ratios. Antibodies induced early after the initial immunization would be expected to be of lower affinity than antibodies present later in the immunization regimen, which may affect estimates of neutralization. For example, Fc receptor-mediated neutralization might be more pronounced for lower-affinity antibodies if an Fc receptor tends to hold the antibody at the cell surface, thereby stabilizing the antibody-PA complex. IgM would also be expected to be more prominent early in the immunization regimen than later, when IgG would be predominant. Such isotype switching might be expected to affect the relative contribution of Fc receptor-mediated neutralization in the J774A.1 cell- and/or RAW 264.7 cell-based assays since Fc receptors are isotype specific. For these reasons, differences in the kinetics of the antibody maturation process in individual animals may contribute to the heterogeneity observed for the RP ratios of sera obtained after a single immunization.
While we obtained comparable estimates of neutralization in the three TNAs when rabbit sera were analyzed, the estimates of neutralization agreed less well when human and NHP sera were analyzed. Moreover, our observation that the J774/CHO as well as the RAW/CHO RP ratios were significantly different for sera from different species suggest that one or more of the TNAs exhibit some species dependence. A possible source for species dependence might be the Fc receptor-dependent neutralization that is known to exist with macrophage-like cells. In support of this explanation, we found that a human serum sample pool exhibited significantly more neutralization that could be blocked by an Fc receptor-blocking monoclonal antibody compared to that of a rabbit serum sample pool in both the J774A.1 and the RAW 264.7 cell assays. We should note that the Fc receptor-blocking antibody used in these studies, MAb 2.4G2, blocks Fc receptor-mediated neutralization, which is specifically dependent on Fcγ receptors IIB and III (29
), and does not necessarily block all Fc receptor-dependent neutralization that may be present in the assays. Nonetheless, the results observed illustrate the possibility that Fc receptor-mediated neutralization could influence estimates of neutralization and may impart some species specificity to certain of the TNAs.
The difference in the contribution of Fc receptor-mediated neutralization observed for the two serum sample pools might be explained by the fact that the Fc regions of human and rabbit antibodies are not identical. This difference in structure could translate into differential binding of the antibodies to the murine Fc receptors present on the macrophage-like cells. These results should be considered when direct comparisons of the neutralization levels of different species are made by TNA, such as will be done when the Animal Rule is applied to new PA-based vaccines.
At this time, the biological relevance of Fc receptor-mediated neutralization is not known. Additional studies will be needed to assess the extent to which Fc receptor-mediated neutralization might play a role in protection against B. anthracis infection in vivo. Such information would be useful when choosing the most appropriate assay format for a given purpose.
Our finding that the three TNAs yielded similar estimates of neutralization when rabbit sera were analyzed, especially after two or three immunizations, but not when either human or NHP sera were analyzed may stem from the fact that the common reference serum sample that we used for each of the three assays was derived from rabbits. This reference serum sample was a polyclonal antiserum sample pooled from rabbits that had received either two or three immunizations of the rPA vaccine. Therefore, the reference serum sample would be expected to behave in a manner most similar to the manners of the rabbit test serum samples in our study that were obtained after either two or three immunizations. The behavior of the reference serum sample is critical and likely dictates whether estimates of neutralization, based on RP values, from the three TNAs will be similar or different. In this regard, our findings serve to emphasize the basic tenet that a good reference sample is one that behaves in a manner similar to the manners of the test samples and further illustrate the importance of the choice of reference sample for use in these types of assays. Our results demonstrate that when the suitability of a reference sample is determined, not only should assay output parameters such as slope, upper asymptote, and lower asymptote be considered—which, for a given assay, were similar between the reference sample and all test serum samples used in this study—but more subtle features of the reference serum sample, such as isotype composition and the species of origin, may also be important considerations, since these characteristics might contribute to the behavior of the reference sample, at least in certain TNA formats. Use of a reference serum sample offers many advantages, such as minimizing assay-to-assay variability and better allowing interlaboratory comparisons. While we believe that these advantages outweigh the potential disadvantages, the findings presented here should be considered when an optimal reference serum sample for use in TNAs is chosen.
The results from this study demonstrate that the Fc receptor-mediated component of neutralization observed in the J774A.1 cell- and RAW 264.7 cell-based assays is associated with some level of species dependence. For many uses of the assays, this may not be a concern. However, for other uses, such as when TNA titers for animals need to be directly compared to those for humans, this finding will be more pertinent. Ultimately, the choice of TNA format that is best for a particular purpose must be regarded in the context of the specific questions that the assay will be used to address. A number of considerations may come into play, some of which involve the biological characteristics of the assay and others of which may be more practical in nature, such as cost and amenability to high-throughput operation.
In conclusion, we have demonstrated that, in general, the level of agreement between the three TNA formats for many purposes is reasonable, but not perfect, at least for the serum samples that were used for the study. The greatest difference in RP estimates observed between the assays was approximately 2.5-fold in magnitude, with some species specificity being noted. Given the differences between the assays in regards to cell type, the specific toxin used, and assay output, as well as the challenges in choosing a common reference serum sample that is suitable for use with all test samples, these results provide reassurance that, regardless of the TNA format used, estimates of neutralization are not dramatically assay dependent. Nonetheless, the differences in estimates of neutralization that were observed in this study and the geneses of these differences should be considered when the TNA output is interpreted.