Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen.

Toxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA), the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses. In contrast, a receptor binding-deficient mutant of PA, which does not bind receptors and only inefficiently enters antigen-presenting cells by macropinocytosis, elicited very poor antibody responses. A chimeric protein consisting of the receptor binding-deficient PA mutant tethered to the binding subunit of cholera toxin, which efficiently enters cells using the cholera toxin receptor rather than the PA receptor, elicited an anti-PA IgG antibody response similar to that elicited by wild-type PA; however, the chimeric protein elicited a poor toxin neutralizing antibody response. Taken together, our results demonstrate that the antigen capture pathway can dictate the magnitudes of the total IgG and toxin neutralizing antibody responses to PA as well as the ratio of the two responses.IMPORTANCE Neutralizing antibodies provide protection against a number of toxin-mediated bacterial diseases by inhibiting toxin action. Therefore, many bacterial vaccines are designed to induce a toxin neutralizing antibody response. We have used protective antigen (PA), the binding component of anthrax toxin, as a model antigen to investigate immune mechanisms important for the induction of robust toxin neutralizing antibody responses. We found that the pathway used by antigen-presenting cells to capture PA dictates the robustness of the neutralizing antibody response to this antigen. These results provide new insights into immune mechanisms that play an important role in the induction of toxin neutralizing antibody responses and may be useful in the design of new vaccines against toxin-mediated bacterial diseases.

Towards replacement of the acellular pertussis vaccine safety test: Comparison of in vitro cytotoxic activity and in vivo activity in mice.

Because of the exquisite sensitivity of the murine histamine sensitization test (HIST) in detecting minute amounts of active pertussis toxin (PTx), this animal-based test has been used to assure the safety of acellular pertussis vaccines in the United States and other countries around the world. Prompted by humane considerations, efforts are underway to find a suitable in vitro replacement assay that has critical attributes comparable to that of the HIST. In this study, we compared the sensitivity of the in vivo HIST with an in vitro Chinese Hamster Ovary (CHO) cell-based assay. Using vaccine samples that had been spiked with PTx, we found that both assays were capable of detecting as little as 4-10 ng of active pertussis toxin per dose of vaccine; thus, the sensitivities of the two assays are comparable. Because the strength of adsorption of PTx to the vaccine adjuvant could change over time, we also used both assays to examine the bioavailability of PTx in spiked vaccine samples that had been stored at 25 °C for 9 weeks, mimicking long term vaccine storage conditions. We found that both assays detected similar amounts of active PTx in these samples, indicating that bioavailability of the toxin in stored samples was similar. Taken together, our results indicate that critical attributes of the HIST are met by the CHO cell assay used in this study and provide proof of concept that the CHO cell assay may be further considered as a replacement for the in vivo HIST.

Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen.

The spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based vaccines. Using a "genetically deamidated" form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation anthrax vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein. We found that loss of the immunogenicity of rPA vaccine was independent of the presence of adjuvant. We assessed the effect of deamidation on the immunodominant neutralizing B-cell epitopes of rPA and found that these epitopes were not significantly affected by deamidation. In order to assess the effect of deamidation on T-cell help for antibody production elicited by rPA vaccine, we examined the ability of the wild-type and genetically deamidated forms of rPA to serve as hapten carriers. We found that when wild-type and genetically deamidated rPA were modified to similar extents with 2,4-dinitrophenyl hapten (DNP) and then used to immunize mice, higher levels of anti-DNP antibodies were elicited by wild-type DNP-rPA than those elicited by the genetically deamidated DNP-rPA, indicating that wild-type rPA elicits more T-cell help than the genetically deamidated form of the protein. These results suggest that a decrease in the ability of deamidated rPA to elicit T-cell help for antibody production is a possible contributor to its lower immunogenicity.

Use of site-directed mutagenesis to model the effects of spontaneous deamidation on the immunogenicity of Bacillus anthracis protective antigen.

Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)--the major component of new-generation anthrax vaccines--affects vaccine immunogenicity, we created a "genetically deamidated" form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.

Analysis of defined combinations of monoclonal antibodies in anthrax toxin neutralization assays and their synergistic action.

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax.

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.

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

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.

Array Biosensor for Toxin Detection: Continued Advances.

The following review focuses on progress made in the last five years with the NRL Array Biosensor, a portable instrument for rapid and simultaneous detection of multiple targets. Since 2003, the Array Biosensor has been automated and miniaturized for operation at the point-of-use. The Array Biosensor has also been used to demonstrate (1) quantitative immunoassays against an expanded number of toxins and toxin indicators in food and clinical fluids, and (2) the efficacy of semi-selective molecules as alternative recognition moieties. Blind trials, with unknown samples in a variety of matrices, have demonstrated the versatility, sensitivity, and reliability of the automated system.

Failure of layer-by-layer multilayers composed of neutravidin-biotin-labeled antibody for sandwich fluoroimmunosensing.

The high-density attachment of active antibodies or other recognition molecules to the capture surface is one of the fundamental processes in route to developing effective biosensors. One method applied frequently to enzymatic sensor systems has been the layer-by-layer assembly of the bioactive surface. Cui et al. [Cui, X., Pei, R., Wang, Z., Yang, F., Ma, Y., Dong, S., Yang, X., 2003. Biosens. Bioelectron. 18, 59-67] extended this concept to immunosensors, where they formed multilayers composed of avidin and biotinylated antibody and reported this construct to be a potent way to form an effective surface for surface plasmon resonance biodetection. We reexamined this concept in an effort to establish a simple method to improve the activity of polystyrene capture surfaces used in sandwich fluoroimmunoassays for the detection of the target, staphylococcal enterotoxin B (SEB). Using multilayers prepared by alternating between NeutrAvidin and either biotinylated mono or polyclonal anti-SEB antibody, we found that virtually all the SEB-binding activity was derived from the final layer added; and that additional layers provided no observable enhancement in fluoroimmunoassay signal strength.

Multiplexed detection of mycotoxins in foods with a regenerable array.

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Nonantibody-based recognition: alternative molecules for detection of pathogens.

Immunoassays have been well established for many years as the cornerstone of detection technologies. These assays are sensitive, selective and, in general, highly resistant to interference from complex sample matrices when compared with nucleic acid-based tests. However, both antibody- and nucleic acid-based detection systems require a priori knowledge of the target and development of specific reagents; multiplexed assays can become increasingly problematic when attempting to detect a plethora of different targets, the identities of which are unknown. In an effort to circumvent many of the limitations inherent in these conventional assays, other recognition reagents are being explored as alternatives, or indeed as adjuncts, to antibodies for pathogen and toxin detection. This article will review a number of different recognition systems ranging in complexity from small molecules, such as nucleic-acid aptamers, carbohydrates and peptides, to systems as highly complicated as whole cells and organisms. All of these alternative systems have tremendous potential to achieve superior sensitivity, selectivity, and stability, but are also subject to their own limitations, which are also discussed. In short, while in its infancy, this field holds great promise for the development of rapid, fieldable assays that are highly complementary to existing antibody- and nucleic acid-based technologies.

An array biosensor for detection of bacterial and toxic contaminants of foods.

The Naval Research Laboratory has developed an array-based biosensor system capable of detecting multiple pathogenic and toxic species in complex matrices. Sandwich fluoroimmunoassays are performed on the surface of a patterned microscope slide that acts as an optical waveguide. Fluorescence from immunocomplexes formed on the slide surface is excited using the evanescent field, an electromagnetic component of light, and the pattern of fluorescence is imaged using a charge-coupled device camera. Using the evanescent wave for excitation allows real-time imaging. Alternatively, a confocal scanner can also be used to detect and quantify fluorescent spots. A method for immobilizing capture antibodies, performing assays, and detecting bound targets is presented.

Detection of deoxynivalenol in foods and indoor air using an array biosensor.

Deoxynivalenol (DON), a mycotoxin produced by several Fusaruim species, is a worldwide contaminant of foods and feeds. Because of the potential dangers due to accidental or intentional contamination of foods with DON, there is a need to develop a rapid and highly sensitive method for easy identification and quantification of DON. In this study, we have developed and utilized a competitive immunoassay technique to detect DON in various food matrixes and indoor air samples using an array biosensor. A DON-biotin conjugate, immobilized on a NeutrAvidin-coated optical waveguide, competed with the DON in the sample for binding to fluorescently labeled DON monoclonal antibodies. To demonstrate a simple procedure amenable for on-site use, DON-spiked cornmeal, cornflakes, wheat, barley, and oats were extracted with methanol-water (3:1) and assayed without cleanup or preconcentration. The limits of detection ranged from 0.2 ng/mL in buffer to 50 ng/g in oats. The detection limit of DON spiked into an aqueous effluent from an air sampler was 4 ng/mL.

Simultaneous determination of kinetic parameters for the binding of cholera toxin to immobilized sialic acid and monoclonal antibody using an array biosensor.

Interactions between protein toxins and carbohydrate receptors are often semi-selective processes and the kinetic parameters that define the binding of a receptor to different toxins may vary with each interaction. In this study, we have determined the affinity constants for binding of cholera toxin (CT) to immobilized sialic acid and to anti-CT antibody (as a simultaneous reference) by measuring real-time binding processes using an array biosensor. N-Acetylneuraminic acid (Neu5Ac), a member of the sialic acid family, was covalently immobilized onto maleimide-activated planar waveguides via a thiol-terminated linker attached to the anomeric carbon of the sugar. Control antibodies were immobilized using two different approaches: covalent attachment onto maleimide-activated slides via the thiol on cysteine residues and non-covalent attachment using a biotin-NeutrAvidin linkage. Cy5-labeled CT was flowed over the immobilized receptors and the fluorescent intensity of the bound CT-receptor complex was recorded as a function of time. The association constants for CT binding to covalently attached Neu5Ac, to covalently attached anti-CT monoclonal antibody, and to antibody tethered by biotin-NeutrAvidin interactions were determined to be 1.3 x 10(8), 2.1 x 10(8) and 5.7 x 10(8)M(-1), respectively.

Detection of bacterial toxins with monosaccharide arrays.

A large number of bacterial toxins, viruses and bacteria target carbohydrate derivatives on the cell surface to attach and gain entry into the cell. We report here the use of a monosaccharide-based array to detect protein toxins. The array-based technique provides the capability to perform simultaneous multianalyte analyses. Arrays of N-acetyl galactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives were immobilized on the surface of a planar waveguide and were used as receptors for protein toxins. These arrays were probed with fluorescently labeled bacterial cells and protein toxins. While Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and staphylococcal enterotoxin B (SEB) did not bind to either of the monosaccharides, both cholera toxin and tetanus toxin bound to GalNAc and Neu5Ac. The results show that the binding of the toxins to the carbohydrates is density dependent and semi-selective. Both toxins were detectable at 100 ng/ml.

Array biosensor for detection of ochratoxin A in cereals and beverages.

Contamination of food by mycotoxins occurs in minute quantities, and therefore, there is a need for a highly sensitive and selective device that can detect and quantify these organic toxins. We report the development of a rapid and highly sensitive array biosensor for the detection and quantitation of ochratoxin A (OTA). The array biosensor utilizes a competitive immunoassay format. Immobilized OTA derivatives compete with toxin in solution for binding to fluorescent anti-OTA antibody spiked into the sample. This competition is quantified by measuring the formation of the fluorescent immunocomplex on the waveguide surface. The fluorescent signal is inversely proportional to the concentration of OTA in the sample. Analyses for OTA in buffer and a variety of food and beverage samples were performed. Samples were extracted with methanol, without any sample cleanup or preconcentration step prior to analysis. The limit of detection for OTA in several cereals ranged from 3.8 to 100 ng/g, while in coffee and wine, detection limits were 7 and 38 ng/g, respectively.