The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.
The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab′)2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines.
The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-Protective Antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to Lethal Factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study (n = 1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.
Bacillus anthracis; Anthrax; Anthrax Vaccine Adsorbed; Lethal Factor; Protective Antigen; correlate of protection
A less than adequate therapeutic plan for the treatment of anthrax in the 2001 bioterrorism attacks has highlighted the importance of developing alternative or complementary therapeutic approaches for biothreat agents. In these regards passive immunization possesses several important advantages over active vaccination and the use of antibiotics, as it can provide immediate protection against Bacillus anthracis. Herein, we report the selection and characterization of several human monoclonal neutralizing antibodies against the toxin of B. anthracis from a phage displayed human scFv library. In total fifteen clones were selected with distinct sequences and high specificity to protective antigen and thus were the subject of a series of both biophysical and cell-based cytotoxicity assays. From this panel of antibodies a set of neutralizing antibodies were identified, of which clone A8 recognizes the lethal (and/or edema) factor binding domain, and clone F1, G11 and G12 recognize the cellular receptor binding domain within protective antigen. It was noted that all clones distinguish a conformational epitope existing on the protective antigen; this steric relationship was uncovered using a sequential epitope mapping approach. For each neutralizing antibody, the kinetic constants were determined by surface plasmon resonance, while the potency of protection was established using a two-tier macrophage cytotoxicity assay. Among the neutralizing antibodies identified, clone F1 possessed the highest affinity to protective antigen, and provided superior protection from lethal toxin in the cell cytotoxicity assay. The data presented provides to the ever-growing arsenal of immunological and functional analysis of monoclonal antibodies to the exotoxins of anthrax. In addition it grants new candidates for the prophylaxis and therapeutic treatment against this toxin.
Bacillus anthracis; protective antigen; human monoclonal antibodies; neutralizing antibodies; phage antibody library
Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD50) and against an aerosol with 75 LD50 of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.
Protective antigen (PA) is the cell surface recognition unit of the binary anthrax toxin system and the primary immunogenic component in both the current and proposed “next-generation” anthrax vaccines. Several studies utilizing animal models have indicated that PA-specific antibodies, acquired by either active or passive immunization, are sufficient to protect against infection with Bacillus anthracis. To investigate the human antibody response to anthrax immunization, we have established a large panel of human PA-specific monoclonal antibodies derived from multiple individuals vaccinated with the currently approved anthrax vaccine BioThrax. We have determined that although these antibodies bind PA in standard binding assays such as enzyme-linked immunosorbent assay, Western blotting, capture assays, and dot blots, less than 25% are capable of neutralizing lethal toxin (LT) in vitro. Nonneutralizing antibodies also fail to neutralize toxin when present in combination with other nonneutralizing paratopes. Although neutralizing antibodies recognize determinants throughout the PA monomer, they are significantly less common among those paratopes that bind to the immunodominant amino-terminal portion of the molecule. These findings demonstrate that PA binding alone is not sufficient to neutralize LT and suggest that for an antibody to effectively block PA-mediated toxicity, it must bind to PA such that one of the requisite toxin functions is disrupted. A vaccine design strategy that directed a higher percentage of the antibody response toward neutralizing epitopes may result in a more efficacious vaccine for the prevention of anthrax infection.
The primary immunogenic component of the currently approved anthrax vaccine is the protective antigen (PA) unit of the binary toxin system. PA-specific antibodies neutralize anthrax toxins and protect against infection. Recent research has determined that in humans, only antibodies specific for particular determinants are capable of effecting toxin neutralization, and that the neutralizing epitopes recognized by these antibodies are distributed throughout the PA monomer. The mechanisms by which the majority of these epitopes effect neutralization remain unknown. In this report we investigate the process by which a human monoclonal antibody specific for the amino-terminal domain of PA neutralizes lethal toxin in an in vitro assay of cytotoxicity, and find that it neutralizes LT by blocking the requisite cleavage of the amino-terminal 20 kD portion of the molecule (PA20) from the remainder of the PA monomer. We also demonstrate that the epitope recognized by this human monoclonal does not encompass the 166RKKR169 furin recognition sequence in domain 1 of PA.
anthrax; Bacillus anthracis; antibody epitope; paratope; human monoclonal antibody; protective antigen; furin; toxin neutralization
A substantial fraction of individuals vaccinated against anthrax have low to immeasurable levels of serum Lethal Toxin (LeTx)-neutralizing activity. The only known correlate of protection against Bacillus anthracis in the currently licensed vaccine is magnitude of the IgG response to Protective Antigen (PA); however, some individuals producing high serum levels of anti-PA IgG fail to neutralize LeTx in vitro. This suggests that non-protective humoral responses to PA may be immunodominant in some individuals. Therefore, to better understand why anthrax vaccination elicits heterogeneous levels of protection, this study was designed to elucidate the relationship between anti-PA fine specificity and LeTx neutralization in response to PA vaccination. Inbred mice immunized with recombinant PA produced high levels of anti-PA IgG and neutralized LeTx in vitro and in vivo. Decapeptide binding studies using pooled sera reproducibly identified the same 9 epitopes. Unexpectedly, sera from individual mice revealed substantial heterogeneity in the anti-PA IgG and LeTx neutralization responses, despite relative genetic homogeneity, shared environment and exposure to the same immunogen. This heterogeneity permitted the identification of specificities that correlate with LeTx-neutralizing activity. IgG binding to six decapeptides comprising two PA epitopes, located in domains I and IV, significantly correlate with seroconversion to LeTx neutralization. These results indicate that stochastic variation in humoral immunity is likely to be a major contributor to the general problem of heterogeneity in vaccine responsiveness and suggest that vaccine effectiveness could be improved by approaches that focus the humoral response toward protective epitopes in a greater fraction of vaccinees.
Bacillus anthracis; Protective antigen; Vaccine; B cell epitope; Mice
The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines.
The bipartite anthrax lethal toxin (LeTx) consisting of protective antigen (PA) and lethal factor (LF) is a major virulence factor contributing to death from systemic Bacillus anthracis infection. The current vaccine elicits antibodies directed primarily to PA; however, in experimental settings serologic responses to LF can neutralize LeTx and contribute to protection against infection. The goals of the present study were to identify sequential B-cell epitopes of LF and to determine the capacity of these determinants to bind neutralizing antibodies. Sera of recombinant LF-immunized A/J mice exhibited high titers of immunoglobulin G anti-LF reactivity that neutralized LeTx in vitro 78 days after the final booster immunization and protected the mice from in vivo challenge with 3 50% lethal doses of LeTx. These sera bound multiple discontinuous epitopes, and there were major clusters of reactivity on native LF. Strikingly, all three neutralizing, LF-specific monoclonal antibodies tested bound specific peptide sequences that coincided with sequential epitopes identified in polyclonal antisera from recombinant LF-immunized mice. This study confirms that LF induces high-titer protective antibodies in vitro and in vivo. Moreover, the binding of short LF peptides by LF-specific neutralizing monoclonal antibodies suggests that generation of protective antibodies by peptide vaccination may be feasible for this antigen. This study paves the way for a more effective anthrax vaccine by identifying discontinuous peptide epitopes of LF.
Prevention or therapy for bioterrorism-associated anthrax infections requires rapidly acting effective vaccines. We recently demonstrated (Y. Tan, N. R. Hackett, J. L. Boyer, and R. G. Crystal, Hum. Gene Ther. 14:1673-1682, 2003) that a single administration of a recombinant serotype 5 adenovirus (Ad) vector expressing anthrax protective antigen (PA) provides rapid protection against anthrax lethal toxin challenge. However, approximately 35 to 50% of humans have preexisting neutralizing antibodies against Ad5. This study assesses the hypothesis that a recombinant adenovirus vaccine based on the nonhuman primate-derived serotype AdC7, against which humans do not have immunity, expressing PA (AdC7PA) will protect against anthrax lethal toxin even in the presence of preexisting anti-Ad5 immunity. Naive and Ad5-immunized BALB/c mice received (intramuscularly) 108 to 1011 particle units (PU) of AdC7PA, Ad5PA (a human serotype Ad5-based vector expressing a secreted form of PA), or AdNull (an Ad5 vector with no transgene). Robust anti-PA immunoglobulin G and neutralizing antibodies were detected by 2 to 4 weeks following administration of AdC7PA to naive or Ad5 preimmunized mice, whereas low anti-PA titers were detected in Ad5-preimmunized mice following administration of Ad5PA. To assess protection in vivo, naive or mice previously immunized against Ad5 were immunized with AdC7PA or Ad5PA and then challenged with a lethal intravenous dose of Bacillus anthracis lethal toxin. Whereas Ad5PA protected naive mice against challenge with B. anthracis lethal toxin, Ad5PA was ineffective in mice that were previously immunized against Ad5. In contrast, AdC7PA functioned effectively not only to protect naive mice but also to protect Ad5-preimmunized mice, with 100% survival after lethal toxin challenge. These data suggest the nonhuman-based vector AdC7PA is an effective vaccine for the development of protective immunity against B. anthracis and importantly functions as a “sero-switch” base for an adenovirus vaccine to function in the context of preexisting anti-Ad immunity.
Anthrax lethal and edema toxins (LeTx and EdTx, respectively) form by binding of lethal factor (LF) or edema factor (EF) to the pore-forming moiety protective antigen (PA). Immunity to LF and EF protects animals from anthrax spore challenge and neutralizes anthrax toxins. The goal of the present study is to identify linear B-cell epitopes of EF and to determine the relative contributions of cross-reactive antibodies of EF and LF to LeTx and EdTx neutralization. A/J mice were immunized with recombinant LF (rLF) or rEF. Pools of LF or EF immune sera were tested for reactivity to rLF or rEF by enzyme-linked immunosorbent assays, in vitro neutralization of LeTx and EdTx, and binding to solid-phase LF and EF decapeptides. Cross-reactive antibodies were isolated by column absorption of EF-binding antibodies from LF immune sera and by column absorption of LF-binding antibodies from EF immune sera. The resulting fractions were subjected to the same assays. Major cross-reactive epitopes were identified as EF amino acids (aa) 257 to 268 and LF aa 265 to 274. Whole LF and EF immune sera neutralized LeTx and EdTx, respectively. However, LF sera did not neutralize EdTx, nor did EF sera neutralize LeTx. Purified cross-reactive immunoglobulin G also failed to cross-neutralize. Cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes. This work increases understanding of the immunogenicity of EF and LF and offers perspective for the development of new strategies for vaccination against anthrax.
Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such as Bacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.
Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including Streptococcus mutans, Porphyromonas gingivalis, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen.
The neutralising anti-PA scFv, 1H, was expressed in L. paracasei as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an in vitro cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, in vivo.
We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both in vitro and in vivo. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.
Bacillus anthracis toxins can be neutralized by antibodies against protective antigen (PA), a component of anthrax toxins. Anthrivig (human anthrax immunoglobulin), also known as AIGIV, derived from plasma of humans immunized with BioThrax (anthrax vaccine adsorbed), is under development for the treatment of toxemia following exposure to anthrax spores. The pharmacokinetics (PK) of AIGIV was assessed in naive animals and healthy human volunteers, and the efficacy of AIGIV was assessed in animals exposed via inhalation to aerosolized B. anthracis spores. In the clinical study, safety, tolerability, and PK were evaluated in three dose cohorts (3.5, 7.1, and 14.2 mg/kg of body weight of anti-PA IgG) with 30 volunteers per cohort. The elimination half-life of AIGIV in rabbits, nonhuman primates (NHPs), and humans following intravenous infusion was estimated to be approximately 4, 12, and 24 days, respectively, and dose proportionality was observed. In a time-based treatment study, AIGIV protected 89 to 100% of animals when administered 12 h postexposure; however, a lower survival rate of 39% was observed when animals were treated 24 h postexposure, underscoring the need for early intervention. In a separate set of studies, animals were treated on an individual basis upon detection of a clinical sign or biomarker of disease, namely, a significant increase in body temperature (SIBT) in rabbits and presence of PA in the serum of NHPs. In these trigger-based intervention studies, AIGIV induced up to 75% survival in rabbits depending on the dose and severity of toxemia at the time of treatment. In NHPs, up to 33% survival was observed in AIGIV-treated animals. (The clinical study has been registered at ClinicalTrials.gov under registration no. NCT00845650.)
The protective effects of polyclonal antisera produced by injecting guinea pigs with protective antigen (PA), the chemical anthrax vaccine AVA, or Sterne spore vaccine, as well as those of toxin-neutralizing monoclonal antibodies (MAbs) produced against PA, lethal factor, and edema factor, were examined in animals infected with Bacillus anthracis spores. Only the anti-PA polyclonal serum significantly protected the guinea pigs from death, with 67% of infected animals surviving. Although none of the MAbs was protective, one PA MAb caused a significant delay in time to death. Our findings demonstrate that antibodies produced against only PA can provide passive protection against anthrax infection in guinea pigs.
The CDC recommend 60 days of oral antibiotics combined with a three-dose series of the anthrax vaccine for prophylaxis after potential exposure to aerosolized Bacillus anthracis spores. The anthrax vaccine is currently not licensed for anthrax postexposure prophylaxis and has to be made available under an Investigational New Drug protocol. Postexposure prophylaxis based on antibiotics can be problematic in cases where the use of antibiotics is contraindicated. Furthermore, there is a concern that an exposure could involve antibiotic-resistant strains of B. anthracis. Availability of alternate treatment modalities that are effective in prophylaxis of inhalation anthrax is therefore highly desirable. A major research focus toward this end has been on passive immunization using polyclonal and monoclonal antibodies against B. anthracis toxin components. Since 2001, significant progress has been made in isolation and commercial development of monoclonal and polyclonal antibodies that function as potent neutralizers of anthrax lethal toxin in both a prophylactic and therapeutic setting. Several new products have completed Phase I clinical trials and are slated for addition to the National Strategic Stockpile. These rapid advances were possible because of major funding made available by the US government through programs such as Bioshield and the Biomedical Advanced Research and Development Authority. Continued government funding is critical to support the development of a robust biodefense industry.
antibiotic treatment; biodefense funding; inhalation anthrax; lethal factor; medical countermeasures; prophylactic antibodies; protective antigen; vaccination
Anthrax is caused by Bacillus anthracis, a gram-positive spore-forming bacterium. Septicemia and toxemia rapidly lead to death in infected mammal hosts. Currently used acellular vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, in experimental animals such vaccines are less protective than live attenuated strains. Here we demonstrate that the addition of formaldehyde-inactivated spores (FIS) of B. anthracis to PA elicits total protection against challenge with virulent B. anthracis strains in mice and guinea pigs. The toxin-neutralizing activities of sera from mice immunized with PA alone or PA plus FIS were similar, suggesting that the protection conferred by PA plus FIS was not only a consequence of the humoral response to PA. A PA-deficient challenge strain was constructed, and its virulence was due solely to its multiplication. Immunization with FIS alone was sufficient to protect mice partially, and guinea pigs totally, against infection with this strain. This suggests that spore antigens contribute to protection. Guinea pigs and mice had very different susceptibilities to infection with the nontoxigenic strain, highlighting the importance of verifying the pertinence of animal models for evaluating anthrax vaccines.
Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. It not only causes natural infection in humans but also poses a great threat as an emerging bioterror agent. The lethality of anthrax is primarily attributed to the two major virulence factors: toxins and capsule. An extensive effort has been made to generate therapeutically useful monoclonal antibodies to each of the virulence components: protective antigen (PA), lethal factor (LF) and edema factor (EF), and the capsule of B. anthracis. This review summarizes the current status of anti-anthrax mAb development and argues for the potential therapeutic advantage of a cocktail of mAbs that recognize different epitopes or different virulence factors.
Bacillus anthracis; anti-PA mAbs; anti-LF mAbs; anti-EF mAbs; anti-capsule mAbs; post-exposure treatment of anthrax; a cocktail of mAbs
The active component of the licensed human anthrax vaccine (BioThrax™, or AVA) is a Bacillus anthracis toxin known as protective antigen (PA). Second generation anthrax vaccines currently under development are also based on a recombinant form of PA. Since the current and future anthrax vaccines are based on this toxin, it is important that the immunobiology of this protein in vaccinated humans be understood in detail. We have isolated and analyzed the PA-specific antibody repertoire from an AVA-vaccinated individual. When examined at the clonal level, we find an antibody response that is complex in terms of the combinatorial elements and immunoglobulin variable genes employed. All PA-specific antibodies had undergone somatic hypermutation and class switch recombination, both signs of affinity maturation. Although the antigenic epitopes recognized by the response were distributed throughout the PA monomer, the majority of antibodies arising in this individual following vaccination recognize determinants located on the amino-terminal (PA20) sub-domain of the molecule. This latter finding may have implications for the rational design of future PA-based anthrax vaccines.
Bacillus anthracis; protective antigen; PA; antibody repertoire; repertoire analysis; anthrax; vaccine; human immune response; human monoclonal antibody
B. anthracis is the causative agent of anthrax. Pathogenesis is primarily mediated through the exotoxins lethal factor and edema factor, which bind protective antigen (PA) to gain entry into the host cell. The current anthrax vaccine (AVA, Biothrax™) consists of aluminum-adsorbed cell-free filtrates of unencapsulated B. anthracis, wherein PA is thought to be the principle target of neutralization. In this study, we evaluated the efficacy of the natural adjuvant, C3d, versus alum in eliciting an anti-PA humoral response and found that C3d conjugation to PA and emulsion in incomplete Freund's adjuvant (IFA) imparted superior protection from anthrax challenge relative to PA in IFA or PA adsorbed to alum. Relative to alum-PA, immunization of mice with C3d-PA/IFA augmented both the onset and sustained production of PA-specific antibodies, including neutralizing antibodies to the receptor-binding portion (domain 4) of PA. C3d-PA/IFA was efficacious when administered either i.p. or s.c., and in adolescent mice lacking a fully mature B cell compartment. Induction of PA-specific antibodies by C3d-PA/IFA correlated with increased efficiency of germinal center formation and plasma cell generation. Importantly, C3d-PA immunization effectively protected mice from intranasal challenge with B. anthracis spores, and was approximately 10-fold more effective than alum-PA immunization or PA/IFA based on dose challenge. These data suggest that incorporation of C3d as an adjuvant may overcome shortcomings of the currently licensed aluminum-based vaccine, and may confer protection in the early days following acute anthrax exposure.
Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.
Neutralizing antibodies to Bacillus anthracis protective antigen (PA), a component of anthrax toxin, mediate protection against anthrax. PA is antigenically complex and can elicit protective and nonprotective antibodies. Furthermore, vaccinated individuals demonstrate considerable variability in their antibody responses to PA. To explore the relationship between PA structure and antigenicity, we produced Escherichia coli strains expressing full-length PA (PA1-4), domains 2 to 4 (PA2-4), domain 1, (PA1), and domain 4 (PA4) and evaluated the immunogenicities and protective efficacies of the protein fractions in four mouse strains (strains A/J, BALB/c, C57BL/6, and Swiss Webster). Immunization with PA1-4 resulted in significantly higher lethal toxin-neutralizing antibody titers than immunization with any recombinant protein (rPA) fraction of PA. The magnitude and neutralizing capacity of the antibody response to full-length PA and its fragments varied depending on the mouse strain. We found no correlation between the antibody titer and the neutralizing antibody titer for A/J and Swiss Webster mice. In C57BL/6 mice, antibody titers and neutralization capacity correlated for two of four rPA domain proteins tested, while BALB/c mice displayed a similar correlation with only one rPA. By correlating the reactivity of immune sera with solvent-exposed linear peptide segments of PA, we tentatively assign the presence of four new linear B-cell epitopes in PA amino acids 121 to 150, 143 to 158, 339 to 359, and 421 to 440. We conclude that the genetic background of the host determines the relative efficacy of the antitoxin response. The results suggest that the variability observed in vaccination studies with PA-derived vaccines is a result of host heterogeneity and implies a need to develop other antigens as vaccine candidates.
The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.
Bacillus anthracis is the causative agent of anthrax, and the tripartite anthrax toxin is an essential element of its pathogenesis. Edema factor (EF), a potent adenylyl cyclase, is one of the toxin components. In this work, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic CB domain. MAb 7F10 shows potent neutralization of edema toxin activity in vitro and in vivo; it targets the N-terminal protective antigen binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor in Western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore-induced foreleg edema model. A combination of three of the MAb also significantly delayed the time to death in a third subcutaneous spore challenge model. This appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression.