Potent anthrax toxin neutralizing human monoclonal antibodies were generated from peripheral blood lymphocytes obtained from Anthrax Vaccine Adsorbed (AVA) immune donors. The anti-anthrax toxin human monoclonal antibodies were evaluated for neutralization of anthrax lethal toxin in vivo in the Fisher 344 rat bolus toxin challenge model.
Human peripheral blood lymphocytes from AVA immunized donors were engrafted into severe combined immunodeficient (SCID) mice. Vaccination with anthrax protective antigen and lethal factor produced a significant increase in antigen specific human IgG in the mouse serum. The antibody producing lymphocytes were immortalized by hybridoma formation. The genes encoding the protective antibodies were rescued and stable cell lines expressing full-length human immunoglobulin were established. The antibodies were characterized by; (1) surface plasmon resonance; (2) inhibition of toxin in an in vitro mouse macrophage cell line protection assay and (3) in vivo in a Fischer 344 bolus lethal toxin challenge model.
The range of antibodies generated were diverse with evidence of extensive hyper mutation, and all were of very high affinity for PA83~1 × 10-10-11M. Moreover all the antibodies were potent inhibitors of anthrax lethal toxin in vitro. A single IV dose of AVP-21D9 or AVP-22G12 was found to confer full protection with as little as 0.5× (AVP-21D9) and 1× (AVP-22G12) molar equivalence relative to the anthrax toxin in the rat challenge prophylaxis model.
Here we describe a powerful technology to capture the recall antibody response to AVA vaccination and provide detailed molecular characterization of the protective human monoclonal antibodies. AVP-21D9, AVP-22G12 and AVP-1C6 protect rats from anthrax lethal toxin at low dose. Aglycosylated versions of the most potent antibodies are also protective in vivo, suggesting that lethal toxin neutralization is not Fc effector mediated. The protective effect of AVP-21D9 persists for at least one week in rats. These potent fully human anti-PA toxin-neutralizing antibodies are attractive candidates for prophylaxis and/or treatment against Anthrax Class A bioterrorism toxins.
A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r2 = 0.89 for log10-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4+ cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.
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
Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.
Bacillus anthracis, the causative agent of anthrax, elaborates a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF) which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin (LT) and edema toxin (ET), respectively. In this preliminary study we characterised the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF with IgG detected as early as 4 days after onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US AVA and UK AVP licensed anthrax vaccines was directed against PA.. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.
anthrax; lethal factor; edema factor; protective antigen
Anthrax Vaccine Adsorbed (AVA) generates short-lived protective antigen (PA) specific IgG that correlates with in vitro toxin neutralization and protection from Bacillus anthracis challenge. Animal studies suggest that when PA-specific IgG has waned, survival after spore challenge correlates with an activation of PA-specific memory B cells. Here, we characterize the quantity and the longevity of AVA-induced memory B cell responses in humans. Peripheral blood mononuclear cells (PBMCs) from individuals vaccinated ≥3 times with AVA (n = 50) were collected early (3–6 months, n = 27) or late after their last vaccination (2–5 years, n = 23), pan-stimulated, and assayed by ELISPOT for total and PA-specific memory B cells differentiated into antibody secreting cells (ASCs). PA-specific ASC percentages ranged from 0.02% to 6.25% (median: 1.57%) and did not differ between early and late post-vaccination individuals. PA-specific ASC percentages correlated with plasma PA-specific IgG (r = 0.42, p = 0.03) and toxin neutralization (r = 0.52, p = 0.003) early post vaccination. PA-specific ASC percentages correlated with supernatant anti-PA both early (r = 0.60, p = 0.001) and late post vaccination (r = 0.71, p < 0.0001). These data suggest PA-specific memory B cell responses are long-lived and can be estimated after recent vaccination by the magnitude and neutralization capacity of the humoral response.
Anthrax Vaccine Adsorbed; cellular immunity; lethal toxin neutralization; protective antigen
Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.
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.
There is a clear need for vaccines and therapeutics for potential biological weapons of mass destruction and emerging diseases. Anthrax, caused by the bacterium Bacillus anthracis, has been used as both a biological warfare agent and bioterrorist weapon previously. Although antibiotic therapy is effective in the early stages of anthrax infection, it does not have any effect once exposed individuals become symptomatic due to B. anthracis exotoxin accumulation. The bipartite exotoxins are the major contributing factors to the morbidity and mortality observed in acute anthrax infections.
Using recombinant B. anthracis protective antigen (PA83), covalently coupled to a novel non-toxic muramyl dipeptide (NT-MDP) derivative we hyper-immunized goats three times over the course of 14 weeks. Goats were plasmapheresed and the IgG fraction (not affinity purified) and F(ab')2 derivatives were characterized in vitro and in vivo for protection against lethal toxin mediated intoxication.
Anti-PA83 IgG conferred 100% protection at 7.5 μg in a cell toxin neutralization assay. Mice exposed to 5 LD50 of Bacillus anthracis Ames spores by intranares inoculation demonstrated 60% survival 14 d post-infection when administered a single bolus dose (32 mg/kg body weight) of anti-PA83 IgG at 24 h post spore challenge. Anti-PA83 F(ab')2 fragments retained similar neutralization and protection levels both in vitro and in vivo.
The protection afforded by these GMP-grade caprine immunotherapeutics post-exposure in the pilot murine model suggests they could be used effectively to treat post-exposure, symptomatic human anthrax patients following a bioterrorism event. These results also indicate that recombinant PA83 coupled to NT-MDP is a potent inducer of neutralizing antibodies and suggest it would be a promising vaccine candidate for anthrax. The ease of production, ease of covalent attachment, and immunostimulatory activity of the NT-MDP indicate it would be a superior adjuvant to alum or other traditional adjuvants in vaccine formulations.
The nontoxic mutant lethal factor (mLF; which has the E687C substitution) and functional protective antigen (PA63) of Bacillus anthracis were evaluated for their use as mucosal vaccines against anthrax in A/J mice. Intranasal vaccination of three doses of 30 μg of mLF or 60 μg of PA63 elicited significant serum and mucosal antibody responses, with anthrax lethal toxin-neutralizing titers of 40 and 60 in immune sera, respectively. However, only 30% and 60% of the vaccinated animals in the two groups could survive a challenge with 100 times the 50% lethal dose of B. anthracis Sterne spores, respectively. In contrast, vaccination with three doses of the combination of 30 μg of mLF and 60 μg of PA63, the detoxified lethal toxin, elicited antibody responses against LF and PA significantly higher than those elicited after vaccination with mLF or PA63 individually by use of the same dose and schedule. Vaccination with the detoxified lethal toxin resulted in significantly higher lethal toxin-neutralizing antibody titers in sera (titer, 90). Animals vaccinated with three doses of the detoxified lethal toxin were completely protected against the spore challenge. The data suggest that mLF and PA63 have a mutual enhancement effect for evoking systemic and mucosal immune responses and that the detoxified lethal toxin can be used as an efficient mucosal vaccine against anthrax.
Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.
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 current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (α-galactosylceramide [α-GC]) at the time of immunization improves PA-specific Ab responses. We observed that α-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d−/− mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jα281−/− mice expressing CD1d but lacking type I α-GC-reactive NKT cells, α-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.
Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA B. anthracis vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA B. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA B. anthracis fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA B. anthracis were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.
Recipients of licensed anthrax vaccine (AVA; Biothrax) could serve as a source of hyperimmune plasma and immunoglobulin for therapy and prophylaxis. We measured serum antibodies during serial weekly to biweekly plasmapheresis in 38 individuals previously vaccinated with 4 to 27 doses of AVA. Immunoglobulin G (IgG) to protective antigen (PA) and toxin neutralization assay (TNA) antibody levels were highly correlated (r = 0.86930 and P < 0.0001 for anti-PA concentration versus TNA concentration). Significant decreases in antibody titer and concentration were observed over time when compared for the number of days from the last AVA injection (P < 0.0001 for both anti-PA and TNA concentration) and for the number of days from the first plasmapheresis (P = 0.0007 for anti-PA concentration and P = 0.0025 for TNA concentration). The rate of the decrease in total IgG concentration (half-life [t1/2] = 198.90 days after first plasmapheresis) was significantly less than the decrease in anti-PA IgG (t1/2 = 63.53 days) (P < 0.0001), indicating that the reduction in anti-PA IgG was more likely due to natural decay than plasmapheresis. The time since the last injection and the time after initial plasmapheresis are important elements in considering an optimal schedule for collecting anthrax hyperimmune plasma. Good correlation between IgG to PA and TNA antibodies suggests that the anti-PA enzyme-linked immunosorbent assay can be used as a high-throughput screen for functional immune reactivity in donor plasma units.
The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with ∼1,000 times the 50% lethal dose (∼1,000× LD50) of B. anthracis Ames strain spores (1.38 × 103 spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10× LD50 and 100× LD50 (1.2 × 106 and 1.2 × 107) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine.
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.
Protective antigen (PA) is a central component of anthrax toxin and a major antigen in anthrax vaccines. However, the use of native PA as a vaccine is not optimal. If administered to people who have been freshly exposed to anthrax, PA may actually aid in anthrax toxin formation and thus may pose a serious safety concern for postexposure vaccination applications. A non-functional PA mutant may be much safer alternative. To identify an improved anthrax vaccine antigen, we examined four non-functional mutants of PA, each being impaired in a critical step of the cellular intoxication pathway of PA. These mutants were Rec- (unable to bind PA-receptors), SSSR (resistant to activation by furin), Oligo- (unable to form oligomers), and DNI (unable to form endosomal transmembrane pores). When tested in mice and after three doses of immunization, all four mutants were highly potent in eliciting PA-specific, toxin-neutralizing antibodies, with immunogenicity increasing in the order of PA < Rec- < SSSR < Oligo- < DNI. While the differences between Rec- or SSSR and PA were small and not statistically significant, DNI and Oligo- were significantly more immunogenic than wild-type PA. One year after immunization and compared with PA-immunized mice, DNI-immunized mice maintained significantly higher levels of anti-PA IgG with correspondingly higher titers of toxin-neutralizing activity. In contrast, Oligo--immunized mice had high levels of anti-PA IgG but lower titers of toxin-neutralizing activity, suggesting that Oligo- mutation sites may overlap with critical protective epitopes of PA. Our study demonstrates that PA-based vaccines could be improved both in terms of safety and efficacy by strategic mutations that not only render PA non-functional but simultaneously enhance its immunogenic potency. Recombinant PA mutants, particularly DNI, hold great promise as better and safer antigens than wild-type PA for use in postexposure vaccination.
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.
Recombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model.
Serological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminumhydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serumantibody titerswere determined by anti-PA IgGELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay.
To examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA.
Neutralizing-antibody titers can be used as a surrogate marker.
anthrax; Bacillus anthracis; Guinea pig; serological correlate
In order to identify the combination of antibody-mediated mechanisms of neutralization that result from vaccination with anthrax vaccine adsorbed (AVA), we isolated antibody secreting cells from a single donor seven days after booster vaccination with AVA and generated nine fully human monoclonal antibodies (hmAb) with high specificity for protective antigen (PA). Two of the antibodies were able to neutralize lethal toxin in vitro at low concentrations (IC50: p6C01, 0.12 µg/ml and p6F01, 0.45 µg/ml). Passive transfer of either of these hmAbs to A/J mice prior to challenge with lethal toxin conferred 80–90% protection. We demonstrate that hmAb p6C01 is neutralizing by preventing furin cleavage of PA in a dose-dependent manner, but the mechanism of p6F01 is unclear. Three additional antibodies were found to bind to domain 3 of PA and prevent oligomerization, although they did not confer significant protection in vivo and showed a significant prozone-like effect in vitro. These fully human antibodies provide insight into the neutralizing response to AVA for future subunit vaccine and passive immunotherapeutic cocktail design.
anthrax; Anthrax Vaccine Adsorbed; human monoclonal antibodies; passive immunotherapeutics; protective antigen
Because of the central role it plays in the formation of lethal toxin and edema toxin, protective antigen (PA) is the principal target for the development of vaccines against anthrax. In the present study, we explored and compared the in vitro and in vivo activities of recombinant anthrax protective antigen (rPA) and receptor binding domain of protective antigen (PA4). As a result, the fully soluble rPA and PA4 proteins were successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx), and the rPA was active in forming cytotoxic lethal toxins, indicating that the rPA protein retains a functionally biological activity. Furthermore, immunization with rPA protein induced stronger PA-specific immune responses in mice than PA4 protein. The protection elicited by immunization with PA4 suggests the presence of common neutralizing epitopes between rPA and PA4, but the immunization with rPA protein induced stronger neutralizing antibodies and protective levels against challenge with the B. anthracis strain A16R than the PA4 protein. The sera neutralizing antibodies titers correlated well with anti-PA group ELISA antibodies titers and the in vivo protective potency. Based on the results of cell cytotoxicity assays and the observed immune responses and protective potency, we concluded that the soluble rPA protein retains the in vitro and in vivo functionally biological activity and can be developed into a highly effective human subunit vaccine candidate against anthrax.
anthrax; PA; activity; immunogenity; vaccine
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.
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.
Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r2 = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD50) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.