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.     

Anthrax: early steps of the intracellular stage of infection development

It was shown that spore germination of different Bacillus anthracis strains in macrophage-like cells J774A.1 depended on the genotype of the strains. The virulent B. anthracis strains contain plasmids pXO1 and pX02 responsible for the synthesis of a toxin and a capsule, respectively. The loss of one of the plasmids results in the reduction of strain virulence. It was shown that effective survival of germinating spores in macrophages occurred in the presence of plasmid pXO1 only. The spores of the B. anthracis strains ?Ames and STI-Rif deprived of plasmid pXO1 were least adapted to passing through the intracellular stage. The B. anthracis strains 81/1 and 71/12 (carrying plasmids pXO1 and pXO2 and synthesizing the toxin and capsule) less effectively survived in the cytoplasm of macrophages than the strain STI-1 which has only the plasmid pXO1. It was found that the rate of synthesis of the capsule consisting of polymer gamma-D-glutamic acid depended on the ability of bacterial cells to escape from macrophages. In the B. anthracis strains carrying plasmid pXO2, capsule synthesis by vegetative cells was activated within macrophages that promoted a rapid escape of the vegetative cells from the macrophages. On the contrary, most of capsule-free cells of the vaccine strain STI-1 remained inside macrophages during the whole period of observation. Thus, integrated regulation of two processes, namely synthesis of the toxin components participating in the transition of the germinating cell from phagosome into cytoplasm, and synthesis of the capsule whose presence promotes rapid escape of bacterial cells from macrophages by presently unknown mechanism play the key role in anthrax development at early stages.     

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。     

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.     

Capsule anchoring in Bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin

Bacillus anthracis , the causative agent of anthrax, is a dangerous biological weapon, as spores derived from drug-resistant strains cause infections for which antibiotic therapy is no longer effective. We sought to develop an anti-infective therapy for anthrax and targeted CapD, an enzyme that cleaves poly-γ- d -glutamate capsule and generates amide bonds with peptidoglycan cross-bridges to deposit capsular material into the envelope of B. anthracis . In agreement with the model that capsule confers protection from phagocytic clearance, B. anthracis capD variants failed to deposit capsule into the envelope and displayed defects in anthrax pathogenesis. By screening chemical libraries, we identified the CapD inhibitor capsidin, 4-[(4-bromophenyl)thio]-3-(diacetylamino)benzoic acid), which covalently modifies the active-site threonine of the transpeptidase. Capsidin treatment blocked capsular assembly by B. anthracis and enabled phagocytic killing of non-encapsulated vegetative forms.     

BslA, a pXO1-encoded adhesin of Bacillus anthracis

The Gram-positive pathogen Bacillus anthracis causes anthrax, a fulminant and lethal infection of mammals. Two large virulence plasmids, pXO1 and pXO2, harbour genes required for anthrax pathogenesis and encode secreted toxins or provide for the poly γ- d -glutamic acid capsule. In addition to capsule, B. anthracis harbours additional cell wall envelope structures, including the surface layer (S-layer), which is composed of crystalline protein arrays. We sought to identify the B. anthracis envelope factor that mediates adherence of vegetative forms to human cells and isolated BslA ( B . anthracis S - l ayer protein A ). Its structural gene, bslA , is located on the pXO1 pathogenicity island (pXO1-90) and bslA expression is both necessary and sufficient for adherence of vegetative forms to host cells. BslA assembly into S-layers and surface exposure is presumably mediated by three N-terminal SLH domains. Twenty-three B. anthracis genes, whose products harbour similar SLH domains, may provide additional surface molecules that allow bacilli to engage cells or tissues of specific hosts during anthrax pathogenesis.     

Protective effect of Bacillus anthracis surface protein EA1 against anthrax in mice

Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA.     

Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease

Bacillus cereus G9241 causes an anthrax-like respiratory illness in humans; however, the molecular mechanisms of disease pathogenesis are not known. Genome sequencing identified two putative virulence plasmids proposed to provide for anthrax toxin (pBCXO1) and/or capsule expression (pBC218). We report here that B. cereus G9241 causes anthrax-like disease in immune-competent mice, which is dependent on each of the two virulence plasmids. pBCXO1 encodes pagA1, the homologue of anthrax protective antigen, as well as hasACB, providing for hyaluronic acid capsule formation, two traits that each contribute to disease pathogenesis. pBC218 harbours bpsX-H, B. cereus exo-polysaccharide, which produce a second capsule. During infection, B. cereus G9241 elaborates both hasACB and bpsX-H capsules, which together are essential for the establishment of anthrax-like disease and the resistance of bacilli to phagocytosis. A single nucleotide deletion causes premature termination of hasA translation in Bacillus anthracis, which is known to escape phagocytic killing by its pXO2 encoded poly-d-γ-glutamic acid (PDGA) capsule. Thus, multiple different gene clusters endow pathogenic bacilli with capsular material, provide for escape from innate host immune responses and aid in establishing the pathogenesis of anthrax-like disease.     

Transposon mutagenesis of Bacillus anthracis strain Sterne using Bursa aurealis

Bacillus anthracis, a spore forming Gram-positive microbe, is the causative agent of anthrax. Although plasmid encoded factors such as lethal toxin (LeTx), edema toxin (EdTx), and gamma-poly-d-glutamic acid (PGA) capsule are known to be required for disease pathogenesis, B. anthracis genes that enable spore invasion, phagosomal escape and macrophage replication are still unknown. To establish transposon mutagenesis as a tool for the characterization of anthrax genes, we employed the mariner-based mini-transposon Bursa aurealis in B. anthracis strain Sterne 7702. B. aurealis carrying an erythromycin resistance cassette and its cognate transposase were delivered by transformation of two plasmids. B. aurealis transposition can be selected for by temperature shift to prevent plasmid replication and by screening colonies for erythromycin resistance. Using inverse polymerase chain reaction, DNA fragments of 129 random erythromycin-resistant transposon mutants were amplified and submitted to DNA sequence analysis. These studies demonstrate that B. aurealis inserts randomly into the genome of B. anthracis and can therefore be employed for finding genes involved in virulence.     

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.     

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.     

Identification of chromosomally encoded membranal polypeptides of Bacillus anthracis by a proteomic analysis: prevalence of proteins containing S-layer homology domains

Bacillus anthracis is the causative agent of anthrax disease. Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells. In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B. anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined. The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF). Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B. anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains. Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein. Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B. anthracis (Vollum strain). This analysis revealed that B. anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response. The identification of the protein components of the B. anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.     

Generation and characterization of monoclonal antibodies to protective antigen of Bacillus anthracis

Monoclonal antibodies (MoAbs) were generated following immunization of BALB/c mice with protective antigen (PA) of B. anthracis. Five clones reactive to this protein were stabilized and preserved. These MoAbs could detect nanogram levels of PA when tested in ELISA. In Western blotting, they reacted with all PA preparations tested and no cross-reactivity was observed with lethal factor, edema factor of B. anthracis and with other organisms. These MoAbs could detect PA from 22 confirmed clinical isolates of B. anthracis on Western blotting and hold promise for direct detection of PA in clinical samples for diagnosing anthrax.     

Genetic diversity in the protective antigen gene of Bacillus anthracis

Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (pag) was sequenced from 26 of the most diverse B. anthracis strains to identify potential variation in the toxin and to further our understanding of B. anthracis evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of pag from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the pag corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in B. anthracis has occurred with little or no horizontal transfer between the different strains.     

Postinfection and postvaccinal antianthrax immunity in human subjects

Antibodies to Bacillus anthracis protective antigen (PA) and to the lethal factor (LF) of B. anthracis exotoxin in the blood sera of anthrax patients and of subjects with a history of the disease, as well as of persons immunized with STI live vaccine, were studied by the heterogeneous enzyme immunoassay. In 1-6 years after convalescence the levels of anti-PA and anti-LF antibodies (at 75% and 96% detection rates respectively) were higher than on weeks 1-4 from the onset of the disease. In persons having had anthrax antibodies belonged mainly to IgG, and the anti-LF antibody level was higher than the anti-PA antibody level. In persons immunized with STI vaccine the detection rate of antibodies somewhat increased in 2-7 months after immunization, reaching, on the average, 72%, the antibody levels after primary immunization and regular annual booster immunization being similar. In 1-2 years after primary (booster) immunization the isolation rate of antibodies decreases to 21%. Specific features of postinfectious and postvaccinal immunity to anthrax and problems of retrospective diagnosis of this disease are discussed.     

In vivo protective role of human group IIa phospholipase A2 against experimental anthrax

Anthrax is an acute disease caused by Bacillus anthracis. Some animal species are relatively resistant to anthrax infection. This trait has been correlated to the extent of the local inflammatory reaction, suggesting innate immunity to be the first line of defense against B. anthracis infection in nonimmunized hosts. Group IIA secreted phospholipase A2 (sPLA2-IIA) is produced in particular by macrophages and possesses potent antibacterial activity especially against Gram-positive bacteria. We have previously shown in vitro that sPLA2-IIA kills both germinated B. anthracis spores and encapsulated bacilli. Here we show that sPLA2-IIA plays in vivo a protective role against experimental anthrax. Transgenic mice expressing human sPLA2-IIA are resistant to B. anthracis infection. In addition, in vivo administration of recombinant human sPLA2-IIA protects mice against B. anthracis infection. The protective effect was observed both with a highly virulent encapsulated nontoxinogenic strain and a wild-type encapsulated toxinogenic strain, showing that toxemia did not hinder the sPLA2-IIA-afforded protection. sPLA2-IIA, a natural component of the immune system, may thus be considered a novel therapeutic agent to be used in adjunct with current therapy for treating anthrax. Its anthracidal activity would be effective even against strains resistant to multiple antibiotics.     

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.     

Antigen delivery by attenuated Bacillus anthracis: new prospects in veterinary vaccines

This report summarizes the recent investigations on the use of Bacillus anthracis as a live vector for delivery of antigens. Recombinant strains were constructed by engineering the current live Sterne vaccine. This vaccine, used to prevent anthrax in cattle, causes side-effects due to anthrax toxin activities. Bacteria producing a genetically detoxified toxin factor were devoid of lethal effects and were as protective as the Sterne strain against experimental anthrax. Moreover, B. anthracis expressing a foreign antigen controlled by an in vivo inducible promoter were able to generate either antibody or cellular protective responses against heterologous diseases.     

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.