Thermostabilization of protective antigen—the binding component of anthrax lethal toxin

Protective antigen (PA) is the binding component of anthrax lethal toxin produced by Bacillus anthracis, and constitutes a major ingredient of the vaccine against anthrax. PA and lethal factor when added together are cytolytic to mouse macrophages and J774G8 macrophage cell line. This in vitro lethal toxicity assay is very useful in understanding the molecular mechanism of action of lethal toxin. Effective utilization of PA is, however, hampered due to its thermolability. On prolonged storage at 37 ° C, PA was found to lose its activity almost completely. The effect of solvent additives like trehalose, sorbitol, xylitol, sodium citrate and magnesium sulphate on the thermal stabilization of PA was examined. The results indicated an increase in the stability of PA when the incubation at 37 ° C was carried out in the presence of solvent additives used in the 1–3 M range. Magnesium sulphate helped retain the activity up to 82.7% against the control in which no additive was used, as judged by cytolytic assay using J774G8 macrophage cell line. Trehalose or sodium citrate also showed an appreciable protection of PA activity, while sorbitol or xylitol were not very effective. Competitive binding assay using radiolabeled PA showed that PA had lost capacity of binding to macrophage cells on prolonged incubation at 37 ° C. Circular dichroism results at 4, 18 and 37 ° C indicated an increase in secondary structure at 37 ° C relative to that at 4 or 18 ° C, supporting the activity data.     

Efficacy of recombinant anthrax vaccine against Bacillus anthracis aerosol spore challenge: Preclinical evaluation in rabbits and Rhesus monkeys

This report describes the immunogenicity and protective efficacy of Escherichia coli-expressed recombinant protective antigen (rPA) in New Zealand White rabbits and Rhesus Macaques against an aerosol challenge with Bacillus anthracis spores (IVRI strain, tox+cap+). A dose-ranging study was performed in which it became evident that the level of anti-PA IgG and toxin-neutralizing antibody titer was directly proportional to the dose of rPA administered. However, the onset time of primary and secondary immune response was not dependent on the dosage. Revaccination of primed animals with the same threshold dose yielded a robust and rapid secondary response. Quantitative differences in peak titers were obtained for both the animal models, in addition to qualitative differences in the immune kinetics. In spite of a weak priming response, the secondary response in rabbits peaked earlier than that in macaques once the booster dose was administered. However, evaluation of the post-challenge quantitative anti-rPA ELISA titer measurements indicated higher titers for non-human primates as compared to the lagomorphs. Importantly, 100% protection was seen for the dosage groups that received ≥25 μg rPA, following a challenge against a target dose of 1000 LD₅₀ of aerosolized spores of Bacillus anthracis.     

Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax

Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.     

炭疽活疫苗家兔免疫力与血清抗芽胞IgG关系的研究

炭疽疫苗是预防炭疽流行和炭疽生物恐怖的重要手段。已有动物实验表明,炭疽活疫苗的保护力优于以保护性抗原为主要成份的无细胞疫苗,但两类现行疫苗都有待重新评价和改进。炭疽疫苗的效力必须用适当的实验室方法进行检测与分析才能了解其性质和细节。试验中力图探寻炭疽活疫苗家兔免疫力与血清抗芽胞抗体水平的关系。用“皮上划痕人用炭疽活疫苗”免疫家兔,以特定制备的炭疽芽胞抗原用ELISA法检测血清抗炭疽芽胞IgG抗体水平,并用强毒炭疽杆菌攻击进行效力试验。免疫家兔血清几何平均抗芽胞IgG滴度在免疫后一个月内持续升高,14d达到206,28d时达到776,这时其抵抗20MLD毒菌攻击的保护率为80％,符合中国生物制品规程要求的保护力。一个月后抗体水平开始下降,42d时滴度降至223。实验所揭示的炭疽减毒活疫苗诱导的家兔抗芽胞IgG抗体与抗炭疽保护力之间的关系,既为评价现行疫苗提供了资料,也为研制新型疫苗建立了参考性指标。     

Lethal factor of anthrax toxin binds monomeric form of protective antigen

Anthrax toxin consists of three components: the enzymatic moieties edema factor (EF) and the lethal factor (LF) and the receptor-binding moiety protective antigen (PA). These toxin components are released from Bacillus anthracis as unassociated proteins and form complexes on the surface of host cells after proteolytic processing of PA into PA20 and PA63. The sequential order of PA heptamerization and ligand binding, as well as the exact mechanism of anthrax toxin entry into cells, are still unclear. In the present study, we provide direct evidence that PA63 monomers are sufficient for binding to the full length LF or its LF-N domain, though with lower affinity with the latter. Therefore, PA oligomerization is not a necessary condition for LF/PA complex formation. In addition, we demonstrated that the PA20 directly interacts with the LF-N domain. Our data points to an alternative process of self-assembly of anthrax toxin on the surface of host cells.     

The 2beta2-2beta3 loop of anthrax protective antigen contains a dominant neutralizing epitope

Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (312)SFFD(315), in the 2beta(2)-2beta(3) loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2beta(2)-2beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.     

Conformational fluctuations in anthrax protective antigen: a possible role of calcium in the folding pathway of the protein

Protective antigen (PA) is the central receptor binding component of anthrax toxin, which translocates catalytic components of the toxin into the cytosol of mammalian cells. Ever since the crystal structure of PA was solved, there have been speculations regarding the possible role of calcium ions present in domain I of the protein. We have carried out a systematic study to elucidate the effect of calcium removal on the structural stability of PA using various optical spectroscopic techniques, limited proteolysis and mutational analysis. Urea denaturation studies clearly suggest that the unfolding pathway of the protein follows a non-two state transition with apo-PA being an intermediate species, whereas the folding pathway shows that calcium ions may be critical for the initial protein assembly.     

针对炭疽保护性抗原不同结构域的中和性单克隆抗体的筛选和鉴定

炭疽保护性抗原（PA）是炭疽毒素的重要组分,同时也是现有炭疽疫苗的主要有效成分,在炭疽杆菌的致病与免疫中发挥关键作用。以重组PA为免疫原,采用B淋巴细胞杂交瘤技术,结合炭疽毒素敏感细胞的毒性中和试验,大量筛选抗PA单克隆抗体,获得了9株炭疽毒素中和性单抗。进一步分析表明这些单抗以IgG1亚类为主,分别识别PA 3个结构域的4个不同中和表位区。针对结构域2的4株单抗识别同一表位区,其中3株单抗的中和活性强于抗PA多抗;针对结构域4的4株单抗识别两个不同表位区;另有1株单抗识别位于结构域3的表位。实验结果提示PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。实验中获得的针对不同表位的中和性单抗为深入研究PA的免疫保护机理提供了工具,也为研制针对炭疽毒素的被动免疫制剂和治疗药物打下基础。     

Exchange characteristics of calcium ions bound to anthrax protective antigen

Protective antigen (PA), the receptor-binding moiety of anthrax toxin, contains two calcium atoms buried within domain 1(') (amino acid residues 168-258). We showed that these ions are stably bound and exchange with free 45Ca(2+) only slowly (t(1/2) approximately 4.0 h). Dissociation is the rate-limiting step. PA(63), the heptameric prepore form of PA, showed a slightly higher exchange rate than the monomeric intact protein. Exchange by this form was retarded by binding of the enzymatic moieties of the toxin, but was unaffected by reducing the pH to 5.0, a condition known to trigger conversion of the prepore to the pore form. These results are consistent with the hypothesis that bound Ca(2+) within PA plays primarily a structural role, maintaining domain 1(') in a conformation that allows PA(63) to oligomerize and bind the enzymatic moieties of the toxin.     

Role of residues constituting the 2beta1 strand of domain II in the biological activity of anthrax protective antigen

Anthrax toxin consists of three proteins, protective antigen, lethal factor and oedema factor. A proteolytically activated 63-kDa fragment of protective antigen binds lethal factor/oedema factor and translocates them into the cytosol. Domain II of protective antigen has been implicated in membrane insertion and channel formation. In the present study, alanine substitutions in 14 consecutive residues of the 2beta1 strand that are highly homologous to the putative membrane interacting segment of Clostridium perfringens iota-b toxin were generated and the effect on the biological activity of protective antigen studied. One of the mutants, Pro260Ala, showed considerably reduced toxicity in combination with lethal factor. The mutant also showed decreased membrane insertion and translocation of lethal factor into the cytosol. The data suggest that Pro260 is important for membrane insertion and translocation by protective antigen.     

Induction of autophagy by anthrax lethal toxin

Autophagy is an evolutionary conserved intracellular process whereby cells break down long-lived proteins and organelles. Accumulating evidences suggest increasing physiological significance of autophagy in pathogenesis of infectious diseases. Anthrax lethal toxin (LT) exerts its influence on numerous cells and herein, we report a novel effect of LT-induced autophagy on mammalian cells. Several autophagy biochemical markers including LC3-II conversion, increased punctuate distribution of GFP-LC3 and development of acidic vesicular organelles (AVO) were detected in cells treated with LT. Analysis of individual LT component revealed a moderate increase in LC3-II conversion for protective antigen-treated cells, whereas the LC3-II level in lethal factor-treated cells remained unchanged. In addition, our preliminary findings suggest a protective role of autophagy in LT intoxication as autophagy inhibition resulted in accelerated cell death. This study presents a hitherto undescribed effect of LT-induced autophagy on cells and provides the groundwork for future studies on the implication of autophagy in anthrax pathogenesis.     

Feasibility of the use of ELISA in an immunogenicity-based potency test of anthrax vaccines

Complexities of lethal challenge animal models have prompted the investigation of immunogenicity assays as potency tests of anthrax vaccines. An ELISA was used to measure the antibody response to protective antigen (PA) in mice immunized once with a commercially available (AVA) or a recombinant PA vaccine (rPAV) formulated in-house with aluminum hydroxide. Results from the anti-PA ELISA were used to select a single dose appropriate for the development of a potency test. Immunization with 0.2 mL of AVA induced a measurable response in the majority of animals. This dose was located in the linear range of the vaccine dose–antibody response curve. In the case of rPAV, practical limitations prevented the finding of the best single dose for the potency testing of purified vaccines. In additional immunogenicity experiments neither the magnitude of the response to a single dose of vaccine, nor the estimation of the dose necessary to induce a measurable response were able to consistently detect brief exposure of vaccines to potentially damaging temperatures. However, differences detected for rPAV in the proportion of mice responding to the same dose of treated and untreated vaccine suggested that further assay development to increase the sensitivity of the latter design may be warranted.     

Effective delivery of antisense peptide nucleic acid oligomers into cells by anthrax protective antigen

Peptide nucleic acid (PNA) is highly stable and binds to complementary RNA and DNA with high affinity, but it resists cellular uptake, thereby limiting its bioavailability. We investigated whether protectiveantigen (PA, a non-toxic component of anthrax toxin) could transport antisense PNA oligomers into reporter cells that contain luciferase transgenes with mutant β-globin IVS2 intronic inserts, which permit aberrant pre-mRNA splicing and impair luciferase expression. PNA oligomers antisense to mutant splice sites in these IVS2 inserts induced luciferase expression when effectively delivered into the cells. PNA 18-mers with C-terminal poly-lysine tails [PNA(Lys)₈] demonstrated modest sequence-specific antisense activity by themselves at micromolar concentrations in luc-IVS2 reporter cell cultures. However, this activity was greatly amplified by PA. Antisense PNA(Lys)₈ with but not without PA also corrected the IVS2-654 β-globin splice defect in cultured erythroid precursor cells from a patient with β-thalassemia [genotype, IVS2-654(β⁰/β^E)], providing further evidence that anthrax PA can effectively transport antisense PNA oligomers into cells.     

Development of a simple and rapid diagnosis method for swine edema disease to specifically detect Stx2e protein by immunochromatographic test

Edema disease in piglets is caused by Shiga toxin 2e (Stx2e)‐producing Escherichia coli. However, there is currently no available Stx2e‐specific immunochromatographic test strip to differentiate Stx2e from other types of Shiga toxin 2. In the present study, to develop an Stx2e‐specific immunochromatographic test strip, we isolated nine different monoclonal antibody‐producing hybridoma clones from Stx2e toxoid‐immunized mice and confirmed that six antibodies were A subunit‐specific whereas three antibodies were B subunit‐specific. Only one A subunit‐specific monoclonal antibody (45B2) was cross‐reactive with prototype Stx2 (Stx2a) at the same sensitivity, but the remaining eight monoclonal antibodies were not. In immunochromatographic tests using the highly sensitive antibodies, test strips using some combinations of gold colloid‐conjugated monoclonal antibody with the B subunit‐specific monoclonal antibody on the membrane detected Stx2e, but not other types of Shiga toxin 2. These test strips had the ability to detect Stx2e in the culture supernatant of clinically isolated Stx2e gene‐positive strains, but not in those of Stx2e gene‐negative strains. These results indicate that our test strip is practical for the specific detection of Stx2e to diagnose swine edema disease.     

Development of a rapid method for the detection of prostate-specific antigen by immunochromatography

A single-step qualitative rapid test for the determination of prostate-specific antigen (PSA) in samples of human blood serum by immunochromatography using a complex of colloidal gold with monoclonal antibodies to PSA as the detection agent was developed. The determination limit for PSA in serum blood samples is 10 ng/ml; the analysis time, 15–25 min; the sensitivity of the method, 100%; and its specificity, 92.5%.     

Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Development of a rapid identification method for Aeromonas species by multiplex-PCR

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.     

The inhibition of the interaction between the anthrax toxin and its cellular receptor by an anti-receptor monoclonal antibody

The high affinity binding of the anthrax protective antigen (PA) to one of its receptors, capillary morphogenesis protein 2 (CMG2), is essential for the intoxication process of anthrax toxin. To acquire novel research tools to study the PA-CMG2 interaction, we generated several anti-CMG2 monoclonal antibodies (MAbs). We demonstrated that one of the MAbs, 4B5, could inhibit PA-CMG2 binding and could also protect the sensitive cells against an anthrax lethal toxin challenge. We identified the epitope recognized by 4B5 and confirmed that the key residues of the epitope were the residues ¹¹⁹YI-LK¹²⁵ of CMG2. Based on our results, we propose that 4B5 binds to the E122 pocket of CMG2 and interrupts the interaction between the pocket and the PA 2β3-2β4 loop. To our knowledge, this is the first report to illustrate that an anti-CMG2 antibody could inhibit the PA-CMG2 interaction and therefore interfere with the intoxication of anthrax toxin.     

Both PA63 and PA83 are endocytosed within an anthrax protective antigen mixed heptamer: a putative mechanism to overcome a furin deficiency

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.