Comparison of ELISA and Toxin Neutralization for the Determination of Tetanus Antibodies

In a sandwich ELISA for tetanus antibodies, the influence of the tetanus toxoid concentration used for coating microtiter plates has been studied. The antibody levels by toxin neutralization bio-assay and by ELISA were studied for a population with known immunization history. By decreasing the tetanus toxoid concentration in ELISA from 5 to 0.2 Lf/ml, a better correlation was found between the ELISA results and the bioassay titers, but sera from recently immunized people still showed high ELISA titers. This phenomenon cannot be ascribed to nonspecific reactions since sera from non-immunized people are negative in both assays. All sera negative in ELISA had, however, a bioassay titer beneath 0.01 IU/ml.     

Combinations of Monoclonal Antibodies to Anthrax Toxin Manifest New Properties in Neutralization Assays

Monoclonal antibodies (MAbs) are potential therapeutic agents against Bacillus anthracis toxins, since there is no current treatment to counteract the detrimental effects of toxemia. In hopes of isolating new protective MAbs to the toxin component lethal factor (LF), we used a strain of mice (C57BL/6) that had not been used in previous studies, generating MAbs to LF. Six LF-binding MAbs were obtained, representing 3 IgG isotypes and one IgM. One MAb (20C1) provided protection from lethal toxin (LeTx) in an in vitro mouse macrophage system but did not provide significant protection in vivo. However, the combination of two MAbs to LF (17F1 and 20C1) provided synergistic increases in protection both in vitro and in vivo. In addition, when these MAbs were mixed with MAbs to protective antigen (PA) previously generated in our laboratory, these MAb combinations produced synergistic toxin neutralization in vitro. But when 17F1 was combined with another MAb to LF, 19C9, the combination resulted in enhanced lethal toxicity. While no single MAb to LF provided significant toxin neutralization, LF-immunized mice were completely protected from infection with B. anthracis strain Sterne, which suggested that a polyclonal response is required for effective toxin neutralization. In total, these studies show that while a single MAb against LeTx may not be effective, combinations of multiple MAbs may provide the most effective form of passive immunotherapy, with the caveat that these may demonstrate emergent properties with regard to protective efficacy.     

Single-Domain Antibodies or Nanobodies: A Class of Next-Generation Antibodies

Nanobodies for the first time were identified in the sera of Camelidae. Single-domain antibodies or nanobodies are a class of next-generation antibodies that have specific features: small size (in nanoscale), high penetration in various tissues, high stability in hard situations and ease production process in microbial systems. In fact single-domain antibodies are the smallest fragment of the antibody with binding ability. Unique characteristics and features of nanobodies make them an appropriate candidate for further evaluation as the development of novel antibody-based therapeutics. In this regard single-domain antibodies are in the interest of many researchers as well as biopharmaceutical companies for diagnostic and therapeutic applications. Nowadays several single domain antibodies have been developed and evaluated in different clinical trials. Because of many advantages of single-domain antibodies over other formats of antibodies, they could be good replacement for other formats of antibodies in near future. Here, we review the biology, engineering platforms and application of nanobodies.     

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 (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ 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 Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ 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 Fcγ 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.Anthrax is a serious and potentially fatal disease caused by Bacillus anthracis. After uptake and germination of anthrax spores within the host, the bacteria elaborate large quantities of anthrax toxin, which is believed to be essential for disease progression (12, 16, 19). The tripartite toxin is composed of a receptor-binding component, protective antigen (PA), and two distinct enzymatic components, lethal factor (LF) and edema factor (16). LF, a Zn²⁺-dependent metalloprotease, combines with PA to form lethal toxin (LT), which is cytotoxic to certain cell types, such as macrophages from susceptible species (2, 6, 7). Edema factor, an adenylate cyclase, combines with PA to form edema toxin, which exerts its effects on cells by dramatically increasing cyclic AMP levels (15).B. anthracis is considered a serious bioterror threat. For this reason, a considerable amount of effort has been devoted to the development of new vaccines and therapeutics for anthrax. While one licensed anthrax vaccine is currently available in the United States, efforts are under way to develop new-generation anthrax vaccines composed of purified recombinant PA (rPA) (9). Antibodies generated by such vaccines would be expected to neutralize the action of both LT and edema toxin and thus protect against disease.The efficacy of new anthrax vaccines cannot be measured directly in humans because such clinical studies are either not feasible or not ethical to conduct. Therefore, in the United States, animal protection data, obtained using two relevant animal species, may serve as primary data to support human efficacy of new-generation anthrax vaccines, as described in the U.S. Government regulation (21 CFR 601, subpart H) commonly known as the “Animal Rule” (3). In order to apply the Animal Rule, animal protection data must be extrapolated to humans in a scientifically sound manner. In the case of anthrax, aerosol infection of nonhuman primates and rabbits is thought to adequately reflect human disease (4, 5). Thus, these two animal models will likely serve as the basis for pivotal animal protection studies. Immunogenicity data will play a critical role in application of the Animal Rule to anthrax vaccines, since serological data from the animals and humans will serve to bridge the animal protective response to human efficacy (5).One serological assay that is likely to be used to assess vaccine immunogenicity in pivotal animal and human studies is the anthrax toxin neutralization assay (TNA), which measures functional antibody levels to anthrax toxin (10, 17). In this assay, the toxin is added to susceptible cells in the presence and absence of neutralizing antibodies. The effect of the toxin on the cells in the presence of antibodies is compared to that observed in their absence. Several forms of the TNA exist, with common forms designed to assess neutralization of the cytotoxic action of LT on either J774A.1 cells or RAW 264.7 cells, both of which are mouse macrophage-like cell lines. The RAW 264.7 cell-based assay has been used primarily in anthrax toxin research (25), whereas the J774A.1 cell-based assay has also been used in clinical vaccine studies and to measure antibody levels in clinical samples (9, 26).Antibody-mediated neutralization in the TNA can occur by “classic” toxin neutralization or, with appropriate cell types, by Fc gamma (Fcγ) receptor-dependent neutralization (31). We define “classic” toxin neutralization as the blocking of a critical step in toxin action, such as receptor binding, pore formation, or enzyme activity. This type of neutralization can be observed with any cell type that is susceptible to the toxin. In contrast, Fcγ receptor-dependent neutralization would be observed only in assays that use cell types that express Fcγ receptors. One such cell type is the macrophage, the cell type that is used for LT neutralization assays because the macrophage, unlike most other cell types, is susceptible to the cytotoxic action of LT. The molecular basis for Fcγ receptor-dependent neutralization is not well understood; however, it may be the result of Fcγ receptor-mediated endocytosis of immune complexes leading to their degradation, sequestration of PA monomers at the cell surface, and/or enhancement of antibody-mediated neutralization by increasing the effective concentration of neutralizing antibodies at the cell surface. Of note, others have observed that nonneutralizing or marginally neutralizing monoclonal antibodies (MAbs) can actually enhance the cytotoxicity of LT on J774A.1 cells through an Fc receptor-dependent mechanism (20). In that case, it was postulated that the Fc receptor-MAb-PA complex decreased the off-rate of PA from the cell surface. Because the antibody did not effectively neutralize PA, stabilization of PA on the cell surface—its target site of action—had the effect of increasing LT toxicity. By similar logic, in the case of neutralizing antibodies, the Fc receptor would be expected to stabilize the antibody at the cell surface, increasing its effective concentration at the very site at which PA accumulates before it combines with LF and exerts its effects on the cell.In order to properly interpret TNA output from pivotal animal and clinical studies and to begin to assess the relevance of the assay output to in vivo protective mechanisms, we need a thorough understanding of the mechanisms of neutralization that contribute to the total neutralization observed in the assay. In this study, we further investigated Fcγ receptor-dependent neutralization in order to better define the molecular basis for this type of toxin neutralization. We also examined the effect of assay parameters, such as cell type and PA concentration, on Fcγ receptor-dependent neutralization in order to better understand the effects that these parameters have on assay output.     

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.     

Characterization of the Antigenic Determinants of Cholera Toxin Subunits

The antigenic specificity of purified preparations of A subunit, B subunit, alpha chain, and gamma chain of cholera toxin was studied by double immunodiffusion and radioimmunoassay with antisera produced in rabbits and mice. Rabbits immunized with A subunit produced serum antibodies which were capable of binding radiolabeled A subunit, alpha chain, and B subunit. Rabbits immunized with alpha chain produced serum antibodies that would bind radiolabeled alpha chain and A subunit. Rabbits immunized with the B subunit produced serum antibodies monospecific for the B subunit. The gamma chain did not elicit measurable antibodies in rabbits or mice as evidenced by radioimmunoassay or double immunodiffusion. A sensitive competitive radioimmunoassay was developed in which the B subunit could inhibit binding of radiolabeled A subunit and alpha chain with either antisera prepared against A subunit or alpha chain. Neither the A subunit nor the alpha chain could inhibit binding of B subunit with the antiserum prepared against B subunit. In addition, selected elution fractions obtained during A- and B-subunit purification were used to immunize groups of mice. Mice responded to immunization with the elution fractions of A subunit by producing anti-A-subunit and anti-B-subunit serum antibody responses, whereas mice immunized with elution fractions of B subunit produced only antibodies specific for the B subunit. An equimolar amount of the two resulting protein peaks was used to immunize two groups of rabbits. Rabbits immunized with A subunit, produced a serum anti-B subunit response equal to that of rabbits immunized with the B subunit. Rabbits immunized with equimolar concentrations of A and B subunits were observed to be equally protected against intestinal loop challenge with Vibrio cholerae Inaba V86. The A subunit, not the B subunit, was biologically active when tested by the S49 mouse lymphosarcoma cell test. These studies provide additional evidence supporting the hypothesis that the A subunit, specifically of alpha chain, of cholera toxin contains antigenic determinants in common with the B subunit.     

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.     

The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization

Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.     

Potent Neutralization of Staphylococcal Enterotoxin B by Synergistic Action of Chimeric Antibodies

Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin''s interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.Staphylococcal enterotoxin B (SEB) is one of several potent exotoxins secreted by Staphylococcus aureus that cause toxic shock syndrome (TSS) (14, 18, 33, 35, 47). This illness, which is characterized by high fever, erythematous rash, and hypotension, can result in multiorgan failure and death. SEB is also a primary cause of classical food poisoning (4). SEB is a superantigen, a category that includes a large number of proteins that can stimulate a large fraction, up to 20%, of the host''s T-cell population (41, 42). Like other superantigens, it binds simultaneously to major histocompatibility complex class II (MHC-II) molecules on antigen-presenting cells (APCs) and to the T-cell receptors (TCRs) that incorporate Vβ chains belonging to particular Vβ families or subfamilies (13, 14, 18, 22, 33). The SEB-induced pathology of TSS results from massive induction of proinflammatory cytokines, which include interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor beta (TNF-β) derived from T_H1 cells (2, 18, 32, 35) and IL-1 and TNF-α from activated APCs (34, 41). Notably, SEB is resistant to denaturation and highly toxic (in humans, the estimated 50% lethal dose is <100 ng/kg of body weight and the 50% effective dose is <1 ng/kg by aerosolized exposure [15, 46]) and can be readily produced by the techniques of recombinant DNA technology. These attributes have led to its classification as a priority B bioterrorism agent.Blockade of SEB''s simultaneous cross-linking of MHC-II on APCs to the TCR on T cells prevents the formation of the MHC-II/SEB/TCR complex and inhibits the action of the toxin. A number of experimental approaches to preventing or disrupting the formation of MHC-II/SAg/TCR complexes have been explored by different laboratories. These include immunization with proteasome-SEB toxoid vaccines (29, 30), inactivated recombinant SEB vaccine (5, 26, 52), and synthetic peptides (53) to induce anti-SEB antibodies, passive immunoprophylaxis and immunotherapy with intravenous immunoglobulin (IVIG) (9, 10, 21, 23), the use of peptide antagonists (1-3), synthetic chimeric mimics of MHC-II/TCR complex (19, 27, 36) or mimics of TCR Vβ (7) engineered to interfere with the binding of SEB to the native forms of these receptors on APCs or T cells. Perhaps the most successful of these approaches have involved TCR Vβ chain mimics that blocked SEB activation in vitro and showed promising results when tested in vivo in a rabbit model (7). However, these TCR mimics reported by Buonpane et al. (7) have a short half-life (325 min) in rabbits and are likely to display short half-lives if deployed in clinical settings. However, rapid in vivo turnover of SEB blocking agents can be avoided by use of antibodies well matched to the host''s FcRn, a receptor responsible for protecting IgG from proteolysis and hence endowing it with a long half-life (24). The use of monoclonal antibodies to neutralize the effects of SEB was first demonstrated by the pioneering studies of Hamad et al. (17) and later by the work of Pang et al. (39). Furthermore, using genes encoding the V regions of monoclonal antibodies derived in nonhuman species, it has been possible to engineer a number of useful chimeric antibodies that manifest relatively long half-lives and low immunogenicity in humans (8). Confident that the V regions of neutralizing mouse monoclonal anti-SEB antibodies could be chimerized with human constant regions, we selected a library of neutralizing anti-SEB from a collection of monoclonal antibodies derived by immunization of BALB/c mice with native SEB.We are also aware that the crystal structures of SEB in complex with MHC-II or TCR reveal that the two binding sites are spatially distinct with the contact areas for each of these different binding sites displaying multiple and potentially immunogenic epitopes against which antibodies can be raised (17). Since multiple epitopes are involved in this interaction, it was possible that our library contained neutralizing antibodies directed against different and spatially distinct epitopes. This suggested that a mixture of anti-SEB antibodies directed against spatially separated neutralizing epitopes would be more effective than an equivalent amount of any component of the mixture used alone. In order to test this hypothesis, it was necessary to identify non-cross-reacting neutralizing antibodies in our library. A pair of non-cross-reactive neutralizing anti-SEB monoclonal mouse antibodies was found and a combination of the two produced a greater degree of neutralization in cultures of mouse splenocytes than equivalent amounts of either member of the pair acting alone. This synergistic action was observed whether the mouse antibodies or chimeric equivalents of the antibody pair were used. However, because it is well established that SEB-mediated effects are seen at much lower toxin concentrations in systems bearing human rather than mouse class II MHC (11, 14), it was important to determine the ability of our pair of chimeric antibodies to neutralize SEB in HLA-DR3 transgenic mice, a more demanding and humanlike model system than conventional mice (11, 44, 45, 50, 51). Both chimeric antibodies effected neutralization in this transgenic model and, when used together, were synergistic in their neutralization of SEB in cultures derived from HLA-DR3 transgenic mice. Similar observations were made in cultures of human peripheral blood mononuclear cells (PBMC).     

Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

The cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.     

Guidelines for Plaque-Reduction Neutralization Testing of Human Antibodies to Dengue Viruses

Through the Advisory Committee on Dengue and other Flavivirus Vaccines, the World Health Organization(WHO) has had a long-standing commitment to facilitate and to guide research and development of vaccines for medically important flaviviruses. Recently, the Paediatric Dengue Vaccine Initiative (PDVI) was formed to accelerate the development, testing, and introduction of dengue (DEN)vaccines worldwide, partnering with WHO in this important public health effort. There are now a variety of DEN vaccines in various stages of the developmental pipeline. In an attempt to make interlaboratory information more directly comparable, WHO with the support of PDVI initiated a program to coordinate the procedures used for the plaque-reduction neutralization test (PRNT). ThePRNT is the most common assay used to measure neutralizing antibody. The presence of antibody is believed to be most relevant means of determining protective anti-DEN virus (DENV) immunity. While other neutralizing antibody assays are being considered for use in large-scale vaccine field trials, the PRNT is still considered to be the laboratory standard against which other neutralizing antibody assays should be compared. The need for PRNT coordination has been identified at several consultations between the WHO and PDVI. A more complete version of these guidelines is available on the WHO website: http://www.who.int/immunization/documents/date/en/index.html.     

Disruption of Anthrax Toxin Binding with the Use of Human Antibodies and Competitive Inhibitors

The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.     

Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.     

Differentiation of Strains of Infectious Bovine Rhinotracheitis Virus by Neutralization Kinetics with Late 19S Rabbit Antibodies

Two vaccine, two respiratory (infectious bovine rhinotracheitis [IBR]), and two genital (infectious pustular vulvovaginitis [IPV]) strains of infectious bovine rhinotracheitis virus were compared by neutralization kinetics using late 19S antibody (AB). The two vaccine strains were indistinguishable from one another, but were neutralized far more rapidly than the other four strains when either anti-IBR or anti-IPV 19S AB was used. The two IPV strains were indistinguishable from one another, but were neutralized significantly more rapidly than the two IBR strains when anti-IBR 19S AB was used. The 2 IBR strains were neutralized at a similar rate with the latter globulin preparation. Almost identical results were obtained with anti-IPV 19S AB, except that one IPV strain was neutralized at a rate similar to the IBR strains. However, when early and late rabbit 7S AB were used, IBR strains could not be distinguished from IPV strains by neutralization kinetics. Preliminary experiments indicated that both early and late 19S rabbit antibodies neutralized the homologous strain more rapidly than the heterologous strain, but the difference was more noticeable with late 19S AB. It was also determined that neutralization of IBR-IPV virus by specific early and late 19S rabbit AB and early 7S rabbit AB was markedly enhanced by guinea pig complement. Neutralization of this virus by late 7S AB, however, was only slightly enhanced by complement. These results suggest that vaccine strains of IBR-IPV virus may be distinguished by neutralization kinetics with late 19S rabbit AB, and that genital and respiratory strains may possibly also be distinguishable with some 19S AB preparations.     

Bispecific Antibodies and Trispecific Immunocytokines for Targeting the Immune System Against Cancer

Monoclonal anti-tumor antibodies (mAbs) that are clinically effective usually recruit, via their constant fragment (Fc) domain, Fc receptor (FcR)-positive accessory cells of the immune system and engage these additionally against the tumor. Since T cells are FcR negative, these important cells are not getting involved. In contrast to mAbs, bispecific antibodies (bsAbs) can be designed in such a way that they involve T cells. bsAbs are artificially designed molecules that bind simultaneously to two different antigens, one on the tumor cell, the other one on an immune effector cell such as CD3 on T cells. Such dual antibody constructs can cross-link tumor cells and T cells. Many such bsAb molecules at the surface of tumor cells can thus build a bridge to T cells and aggregate their CD3 molecules, thereby activating them for cytotoxic activity. BsAbs can also contain a third binding site, for instance a Fc domain or a cytokine that would bind to its respective cytokine receptor. The present review discusses the pros and cons for the use of the Fc fragment during the development of bsAbs using either cell-fusion or recombinant DNA technologies. The recombinant antibody technology allows the generation of very efficient bsAbs containing no Fc domain such as the bi-specific T-cell engager (BiTE). The strong antitumor activity of these molecules makes them very interesting new cancer therapeutics. Over the last decade, we have developed another concept, namely to combine bsAbs and multivalent immunocytokines with a tumor cell vaccine. The latter are patient-derived tumor cells modified by infection with a virus. The virus—Newcastle Disease Virus (NDV)—introduces, at the surface of the tumor cells, viral molecules that can serve as general anchors for the bsAbs. Our strategy aims at redirecting, in an Fc-independent fashion, activities of T cells and accessory cells against autologous tumor antigens. It creates very promising perspectives for a new generation of efficient and safe cancer therapeutics that should confer long-lasting anti-tumor immunity.     

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A2 and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A₁ peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A₂ peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2ΔAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A₂ sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2ΔAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens representing the major etiological agents of hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease characterized by hemolytic anemia, thrombocytopenia, and renal failure (19). The infection correlates with ingestion of contaminated meat or vegetables but is also transmitted by water or even person-to-person contact (8, 14, 44). Sporadic or massive outbreaks have been reported in several developed countries but, in Argentina, HUS is endemic and represents a serious public health problem with high morbidity and mortality rates (29, 40). Production of verocytotoxin or Shiga-like toxin (Stx) is the basis of EHEC pathogenesis (18, 20). The toxin is formed by a single A subunit, which possesses N-glycosidase activity to the 28S rRNA and promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells (9, 28). Although two major types (Stx1 and Stx2) and several subtypes have been described, Stx2 and Stx2c are the most frequently found toxins in severe HUS cases among EHEC-infected subjects (12, 41). The degree of antigenic cross-reactivity between Stx2 and Stx1 is low, and several authors have reported that the two toxins do not provide heterologous protection, particularly concerning the B subunits (45, 47). On the other hand, Stx2c and Stx2d variants are readily neutralized by antibodies against Stx2 (27).Despite the magnitude of the social and economic impacts caused by EHEC infections, no licensed vaccine or effective therapy is presently available for human use. So far, attempts to develop vaccine formulations against EHEC-associated sequelae have relied mainly on induction of serum anti-Stx antibody responses. Several approaches have been pursed to generate immunogenic anti-Stx vaccine formulations and include the use of live attenuated bacterial strains (2, 32), protein-conjugated polysaccharides (21), purified B subunit (33, 48), B-subunit-derived synthetic peptides (15), and mutated Stx1 and Stx2 nontoxic derivatives (5, 6, 13, 16, 37, 39, 42, 45).In a previous report we described anti-Stx2 DNA vaccines encoding either the B subunit or a fusion protein between the B subunit and the first N-terminal amino acid of the A₁ subunit (8). The DNA vaccine encoding the hybrid protein elicited Stx-specific immune responses in mice and partial protection to Stx2 challenge (1, 33). Recent data have indicated that epitopes leading to generation of Stx-neutralizing antibodies are present on both the B as well as the A subunit (34, 45, 46). In addition, further evidence indicates that the A₂ subunit contains some of the most immunogenic epitopes of the Stx2 toxin (4). Thus, in line with such evidence, we attempted the construction of a new DNA vaccine encoding the last 32 amino acids from the A₂ subunit, in addition to the complete B subunit of Stx2, as separated polypeptides which could enhance the immunogenicity and protective effects of the vaccine formulation. In the present report, we describe the generation of a new DNA vaccine encoding both Stx2 A2 and B subunits as an approach to elicit protective antibody responses to Stx2. The results obtained demonstrate that immunization with this vaccine formulation results in systemic antibody responses to Stx2 A and B subunits and toxin neutralization activity both in vitro and in vivo.