Recent progress in the development of anthrax vaccines

Bacillus anthracis is the etiological agent of anthrax. Although anthrax is primarily an epizootic disease; humans are at risk for contracting anthrax. The potential use of B. anthracis spores as biowarfare agent has led to immense attention. Prolonged vaccination schedule of current anthrax vaccine and variable protection conferred; often leading to failure of therapy. This highlights the need for alternative anthrax countermeasures. A number of approaches are being investigated to substitute or supplement the existing anthrax vaccines. These relied on expression of Protective antigen (PA), the key protective immunogen; in bacterial or plant systems; or utilization of attenuated strains of B. anthracis for immunization. Few studies have established potential of domain IV of PA for immunization. Other targets including the spore, capsule, S-layer and anthrax toxin components have been investigated for imparting protective immunity. It has been shown that co-immunization of PA with domain I of lethal factor that binds PA resulted in higher antibody responses. Of the epitope based vaccines, the loop neutralizing determinant, in particular; elicited robust neutralizing antibody response and conferred 97% protection upon challenge. DNA vaccination resulted in varying degree of protection and seems a promising approach. Additionally, the applicability of monoclonal and therapeutic antibodies in the treatment of anthrax has also been demonstrated. The recent progress in the direction of anthrax prophylaxis has been evaluated in this review.     

Poly-γ-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis . The two major virulence factors of B. anthracis are exotoxin and the poly-γ- d -glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA–PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 × LD₅₀ challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA–PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.     

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.     

Complement C3d conjugation to anthrax protective antigen promotes a rapid, sustained, and protective antibody response

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.     

利用转基因小鼠筛选全人源炭疽致死因子中和抗体

炭疽是由炭疽芽孢杆菌引起的严重威胁人类健康的传染病。炭疽毒素包括3种蛋白质成分:保护性抗原(PA)、致死因子(LF)和水肿因子(EF)。PA与LF形成致死毒素(LT),与EF形成水肿毒素(ET)。由于致死毒素(LT)在感染者损伤及死亡中发挥主要作用,因此在炭疽感染晚期单纯使用抗生素治疗难以发挥疗效,治疗性中和抗体成为目前最有效的炭疽治疗药物。目前国外获得的炭疽毒素抗体多为炭疽PA抗体,美国FDA已批准瑞西巴库(人源PA单抗)用于吸入性炭疽的治疗。一旦炭疽芽孢杆菌被人为改构或PA中和表位发生突变,针对PA单一表位的抗体将可能失效,因此针对LF的抗体将成为炭疽治疗的有效补充。目前国外已有的LF抗体多为鼠源抗体和嵌合抗体,而全人源抗体可以避免鼠源抗体免疫原性高等缺点。本研究首先用LF抗原免疫人抗体转基因小鼠,利用流式细胞仪从小鼠脾淋巴细胞中分选抗原特异的记忆B细胞,通过单细胞PCR方法快速获得两株具有结合活性的抗LF单抗1D7和2B9。瞬时转染Expi 293F细胞制备抗体,通过毒素中和实验(TNA)发现1D7和2B9在细胞模型中均显示较好的中和活性,并且与PA单抗联合使用时,表现出较好的协同作用。总之,本文利用转基因小鼠、流式分选技术和单细胞PCR技术的优势,快速筛选到全人源LF抗体,为快速筛选全人源单克隆抗体开辟了新的思路与方法。     

Nasal immunization with the mixture of PA63, LF, and a PGA conjugate induced strong antibody responses against all three antigens

A new generation anthrax vaccine is expected to target not only the anthrax protective antigen (PA) protein, but also other virulent factors of Bacillus anthracis. It is also expected to be amenable for rapid mass immunization of a large number of people. This study aimed to address these needs by designing a prototypic triantigen nasal anthrax vaccine candidate that contained a truncated PA (rPA63), the anthrax lethal factor (LF), and the capsular poly-gamma-D-glutamic acid (gammaDPGA) as the antigens and a synthetic double-stranded RNA (dsRNA), polyriboinosinic-polyribocytodylic acid (poly(I:C)) as the adjuvant. This study identified the optimal dose of nasal poly(I:C) in mice, demonstrated that nasal immunization of mice with the LF was capable of inducing functional anti-LF antibodies (Abs), and showed that nasal immunization of mice with the prototypic triantigen vaccine candidate induced strong immune responses against all three antigens. The immune responses protected macrophages against an anthrax lethal toxin challenge in vitro and enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge in vivo. The anti-PGA Abs were shown to have complement-mediated bacteriolytic activity. After further optimization, this triantigen nasal vaccine candidate is expected to become one of the newer generation anthrax vaccines.     

Immunogenicity and efficacy of an anthrax/plague DNA fusion vaccine in a mouse model

The efficacy of multi-agent DNA vaccines consisting of a truncated gene encoding Bacillus anthracis lethal factor (LFn) fused to either Yersinia pestis V antigen (V) or Y .?pestis F1 was evaluated. A/J mice were immunized by gene gun and developed predominantly IgG1 responses that were fully protective against a lethal aerosolized B.?anthracis spore challenge but required the presence of an additional DNA vaccine expressing anthrax protective antigen to boost survival against aerosolized Y.?pestis.     

Retrocyclins kill bacilli and germinating spores of Bacillus anthracis and inactivate anthrax lethal toxin

Theta-defensins are cyclic octadecapeptides encoded by the modified alpha-defensin genes of certain nonhuman primates. The recent demonstration that human alpha-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of theta-defensins on Bacillus anthracis (Sterne). We tested rhesus theta-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of theta-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of theta-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that theta-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.     

Immunogenicity of the recombinant Bacillus strains with cloned gene of biosynthesis of protective antigen against Bacillus anthracis

Microbe Russian Anti-Plague Research Institute, Saratov A hybrid plasmid pUB110PA-1 demonstrating stable functioning in the cells of Bacillus strains and containing the gene of biosynthesis of Bacillus anthracis protective antigen was constructed. The recombinant strains surpassing the anthrax vaccinal cultures in the secreted synthesis of the protective antigen were obtained and their immunological efficacy was assessed. A single inoculation of Guinea pigs with the dose of 5 x 107 spores of the recombinant strains imparted efficient protection against B. anthracis challenge. Immune responses were characterized by high indices of immunity and titers of antibodies to the protective antigen. In contrast to the anthrax vaccinal preparations, the gene-engineering strains imposed no residual virulence for BALB/n mice and Guinea pigs.     

Construction and characterization of a protective antigen-deficient Bacillus anthracis strain

The pag gene coding for protective antigen (PA), one of the three toxin components of Bacillus anthracis, has been cloned into the mobilizable shuttle vector pAT187 and transferred by conjugation from Escherichia coli to B. anthracis. Using this strategy, an insertionally mutated pag gene constructed and characterized in E. coli, was introduced into B. anthracis Sterne strain. This transconjugant was used to select a recombinant clone (RP8) carrying the inactivated pag gene on the toxin-encoding plasmid, pXO1. Strain RP8 was deficient for PA while still producing the two other toxin components, i.e. lethal factor (LF) and edema factor (EF). In contrast to spores from the wild-type Sterne strain, spores prepared from RP8 were totally non-lethal in mice. These results clearly establish the central role played by PA in B. anthracis pathogenicity.     

The early humoral immune response to Bacillus anthracis toxins in patients infected with cutaneous anthrax

Bacillus anthracis, the causative agent of anthrax, produces 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 and edema toxin, respectively. In this preliminary study, we characterized 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 immunoglobulin G (IgG) detected as early as 4 days after the 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 anthrax vaccine absorbed and UK anthrax vaccine precipitated 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.     

Immunosuppressive action of the lethal toxin of Bacillus anthracis in mice

In experiments on inbred mice infected with B. anthracis capsular strain 71/12 of Tsenkovsky's second vaccine B. anthracis lethal toxin introduced in mixture with spores has been shown to aggravate anthrax infection in CBA mice susceptible to anthrax, while producing a faint effect on the infectious process in BALB mice with hereditary resistance to anthrax. B. anthracis purified edema toxin has been found to produce a weaker aggravating effect with respect to anthrax infection than the lethal toxin. As revealed in these experiments, the capacity of the lethal toxin to suppress the activity of peritoneal macrophages in vitro is the more pronounced, the more resistant to anthrax are the mice used as the donors of these macrophages. The mechanism of hereditary immunity which may ensure resistance to infection in the presence of immunosuppression is discussed.     

Immune correlates of protection against anthrax

Bacillus anthracis protective antigen (PA) has been produced from a recombinant B. subtilis and its efficacy, when combined with the Ribi adjuvant (MPL-TDW-CWS) or alhydrogel, has been compared with that of the licensed UK human vaccine, in guinea pigs challenged with aerosolized Ames strain spores. Recombinant PA combined with the Ribi adjuvant performed as well as PA from B. anthracis cultures in previous reports ( Ivins & Welkos 1986 ; Ivins et al . 1990 ; Turnbull et al . 1991 ; Jones et al . 1996 ; McBride et al . 1998 ) giving protection in 100% of animals exposed to the highest challenge dose of the Ames strain of B. anthracis that can be administered practically (retained lung doses of approximately 10⁶ spores).
In attempts at identifying markers of protection in immunized individuals, rPA in combination with the Ribi adjuvant induced a marker IgG₂ response in guinea pigs with no significant differences in IgG₁ levels when compared with other vaccine formulations ( McBride et al . 1998 ). In BALBc mice, rPA with the Ribi adjuvant induced a higher IgG_2a response compared with rPA with anhydrogel and the human vaccine.
To examine the role of anti-PA-specific antibodies in protection, guinea pig sera is being passively transferred into guinea pigs and SCID mice, followed by protection.
Similarly, B- and T-lymphocytes from immunized BALB/c mice are being separately and passively transferred into SCID mice with subsequent challenge. The neutralizing ability of the PA-specific antibodies is being studied using an in vitro macrophage lysis assay.     

Early interactions between fully virulent Bacillus anthracis and macrophages that influence the balance between spore clearance and development of a lethal infection

The role of macrophages in the pathogenesis of anthrax is unresolved. Macrophages are believed to support the initiation of infection by Bacillus anthracis spores, yet are also sporicidal. Furthermore, it is believed that the anthrax toxins suppress normal macrophage function. However, the significance of toxin effects on macrophages has not been addressed in an in vivo infection model. We used mutant derivatives of murine macrophage RAW264.7 cells that are toxin receptor-negative (R3D) to test the role of toxin-targeting of macrophages during a challenge with spores of the Ames strain of B. anthracis in both in vivo and in vitro models. We found that the R3D cells were able to control challenge with Ames when mice were inoculated with the cells prior to spore challenge. These findings were confirmed in vitro by high dose spore infection of macrophages. Interestingly, whereas the R3D cells provided a significantly greater survival advantage against spores than did the wild type RAW264.7 cells or R3D-complemented cells, the protection afforded the mutant and wild type cells was equivalent against a bacillus challenge. The findings appear to be the first specific test of the role of toxin targeting of macrophages during infection with B. anthracis spores.     

Deletion mutants of protective antigen that inhibit anthrax toxin both in vitro and in vivo

The anthrax toxin complex is primarily responsible for most of the symptoms of anthrax. This complex is composed of three proteins, anthrax protective antigen, anthrax edema factor, and anthrax lethal factor. The three proteins act in binary combination of protective antigen plus edema factor (edema toxin) and protective antigen plus lethal factor (lethal toxin) that paralyze the host defenses and eventually kill the host. Both edema factor and lethal factor are intracellularly acting proteins that require protective antigen for their delivery into the host cell. In this study, we show that deletion of certain residues of protective antigen results in variants of protective antigen that inhibit the action of anthrax toxin both in vitro and in vivo. These mutants protected mice against both lethal toxin and edema toxin challenge, even when injected at a 1:8 ratio relative to the wild-type protein. Thus, these mutant proteins are promising candidates that may be used to neutralize the action of anthrax toxin.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge

Initiation of inhalation anthrax is believed to involve phagocytosis of Bacillus anthracis spores by alveolar macrophages, followed by spore germination within the phagolysosome. In order to establish a systemic infection, it is predicted that bacilli then escape from the macrophage and replicate extracellularly. Mechanisms utilized by B. anthracis to escape from the macrophage are not well characterized, but a role for anthrax toxin has been proposed. Here we report the isolation of an anthrax toxin-resistant cell line (R3D) following chemical mutagenesis of toxin-sensitive RAW 264.7 murine macrophage cells. Both R3D and RAW 264.7 cells phagocytize spores of a B. anthracis Sterne strain. However, RAW 264.7 cells are killed following spore challenge, whereas R3D cells survive. Resistance to toxin and spore challenge correlates with loss of expression of anthrax toxin receptor 2 (ANTXR2/CMG-2). When R3D cells are complemented with cDNA encoding either murine ANTXR2 or human anthrax toxin receptor 1 (ANTXR1/TEM-8), toxin and spore challenge susceptibility are restored, indicating that over-expression of either ANTXR can confer susceptibility to anthrax spore challenge. Taken together, these results indicate that anthrax toxin expression by the germinated spore enables B. anthracis killing of the macrophage from within.     

Characterisation of the immune response to the UK human anthrax vaccine

The UK human anthrax vaccine consists of the alum-precipitated culture supernatant of Bacillus anthracis Sterne. In addition to protective antigen (PA), the key immunogen, the vaccine also contains a number of other bacteria- and media-derived proteins. These proteins may contribute to the transient side effects experienced by some individuals and could influence the development of the PA-specific immune response. Bacterial cell-wall components have been shown to be potent immunomodulators. B. anthracis expresses two S-layer proteins, EA1 and Sap, which have been demonstrated to be immunogenic in animal studies. These are also immunogenic in man so that convalescent and post-immunisation sera contain specific antibodies to Ea1, and to a lesser extent, to Sap. To determine if these proteins are capable of modifying the protective immune response to PA, A/J mice were immunised with equivalent amounts of recombinant PA and S-layer proteins in the presence of alhydrogel. IgG isotype profiles were determined and the animals were subsequently challenged with spores of B. anthracis STI. The results suggest that there was no significant shift in IgG isotype profile and that the presence of the S-layer proteins did not adversely affect the protective immune response induced by PA.     

A Single-Dose PLGA Encapsulated Protective Antigen Domain 4 Nanoformulation Protects Mice against Bacillus anthracis Spore Challenge

Bacillus anthracis, the etiological agent of anthrax, is a major bioterror agent. Vaccination is the most effective prophylactic measure available against anthrax. Currently available anthrax vaccines have issues of the multiple booster dose requirement, adjuvant-associated side effects and stability. Use of biocompatible and biodegradable nanoparticles to deliver the antigens to immune cells could solve the issues associated with anthrax vaccines. We hypothesized that the delivery of a stable immunogenic domain 4 of protective antigen (PAD4) of Bacillus anthracis encapsulated in a poly (lactide-co-glycolide) (PLGA) - an FDA approved biocompatible and biodegradable material, may alleviate the problems of booster dose, adjuvant toxicity and stability associated with anthrax vaccines. We made a PLGA based protective antigen domain 4 nanoparticle (PAD4-NP) formulation using water/oil/water solvent evaporation method. Nanoparticles were characterized for antigen content, morphology, size, polydispersity and zeta potential. The immune correlates and protective efficacy of the nanoparticle formulation was evaluated in Swiss Webster outbred mice. Mice were immunized with single dose of PAD4-NP or recombinant PAD4. The PAD4-NP elicited a robust IgG response with mixed IgG1 and IgG2a subtypes, whereas the control PAD4 immunized mice elicited low IgG response with predominant IgG1 subtype. The PAD4-NP generated mixed Th1/Th2 response, whereas PAD4 elicited predominantly Th2 response. When we compared the efficacy of this single-dose vaccine nanoformulation PAD4-NP with that of the recombinant PAD4 in providing protective immunity against a lethal challenge with Bacillus anthracis spores, the median survival of PAD4-NP immunized mice was 6 days as compared to 1 day for PAD4 immunized mice (p<0.001). Thus, we demonstrate, for the first time, the possibility of the development of a single-dose and adjuvant-free protective antigen based anthrax vaccine in the form of PAD4-NP. Further work in this direction may produce a better and safer candidate anthrax vaccine.     

Anthrax toxin targeting of myeloid cells through the CMG2 receptor is essential for establishment of Bacillus anthracis infections in mice

Bacillus anthracis kills through a combination of bacterial infection and toxemia. Anthrax toxin working via the CMG2 receptor mediates lethality late in infection, but its roles early in infection remain unclear. We generated myeloid-lineage specific CMG2-deficient mice to examine the roles of macrophages, neutrophils, and other myeloid cells in anthrax pathogenesis. Macrophages and neutrophils isolated from these mice were resistant to anthrax toxin. However, the myeloid-specific CMG2-deficient mice remained fully sensitive to both anthrax lethal and edema toxins, demonstrating that targeting of myeloid cells is not responsible for anthrax toxin-induced lethality. Surprisingly, the myeloid-specific CMG2-deficient mice were completely resistant to B. anthracis infection. Neutrophil depletion experiments suggest that B. anthracis relies on anthrax toxin secretion to evade the scavenging functions of neutrophils to successfully establish infection. This work demonstrates that anthrax toxin uptake through CMG2 and the resulting impairment of myeloid cells are essential to anthrax infection.