Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 ؋ 10 7 spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>100 g/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.The etiological agent of anthrax disease in animals and humans is the spore-forming bacterium Bacillus anthracis. The major factors of virulence of B. anthracis are located on two plasmids, pXO1 and pXO2. pXO2 encodes a poly-D-glutamic acid capsule (19, 41), while pXO1 encodes two binary exotoxins, the lethal toxin (LT) and the edema toxin (ET) (43,46,61). These two toxins are composed of three different proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF) (for a review, see reference 36). PA is the common receptor binding domain of the toxins and can interact with the two different effector domains, EF and LF, to mediate their entry into target cells (14). EF is a calmodulin-dependent adenylate cyclase (37) responsible for the edema seen at the site of infection in experimental animals (17). The LF is a metalloprotease (34) recently shown to cleave the amino termini of the mitogen-activated protein kinase kinases 1 and 2, which results in their inactivation (13). It remains to be determined whether these are the main physiological substrates for the LT activity in vivo (5,22).Two types of anthrax vaccines are licensed for use in humans: the spores of the toxigenic, nonencapsulated B. anthracis STI-1 strain (55) and the cell-free PA-based vaccines consisting of aluminum hydroxide-adsorbed supernatant material from cultures of the toxigenic, nonencapsulated B. anthracis strain V770-NPI-R (49) or alum-precipitated culture filtrate from the Sterne strain (6). The use of the live attenuated STI-1 occasionally results in general and local adverse responses, observed both after primary application and revaccination, and the frequency of responses increases with the number of vaccinations (58). Furthermore, it was reported that the STI-1 vaccine has a relatively low immunogenicity (reviewed by Stepanov et al. in reference 58). To increase the i...
SummaryHere we describe the characterization of a lipoprotein previously proposed as a potential Bacillus anthracis virulence determinant and vaccine candidate. This protein, designated MntA, is the solute-binding component of a manganese ion ATP-binding cassette transporter. Coupled proteomic-serological screen of a fully virulent wild-type B. anthracis Vollum strain, confirmed that MntA is expressed both in vitro and during infection. Expression of MntA is shown to be independent of the virulence plasmids pXO1 and pXO2. An mnt A deletion, generated by allelic replacement, results in complete loss of MntA expression and its phenotypic analysis revealed: (i) impaired growth in rich media, alleviated by manganese supplementation; (ii) increased sensitivity to oxidative stress; and (iii) delayed release from cultured macrophages. The Δ Δ Δ Δ mnt A mutant expresses the anthraxassociated classical virulence factors, lethal toxin and capsule, in vitro as well as in vivo , and yet the mutation resulted in severe attenuation; a 10 4 -fold drop in LD 50 in a guinea pig model. MntA expressed in trans allowed to restore, almost completely, the virulence of the Δ Δ Δ Δ mnt A B. anthracis strain. We propose that MntA is a novel B. anthracis virulence determinant essential for the development of anthrax disease, and that B. anthracis Δ Δ Δ Δ mnt A strains have the potential to serve as platform for future live attenuated vaccines.
The bovine acetylcholinesterase (BoAChE) gene was cloned from genomic DNA and its structure was determined. Five exons coding for the AChE T-subunit and the alternative H-subunit were identified and their organization suggests high conservation of structure in mammalian AChE genes. The deduced amino acid sequence of the bovine T-subunit is highly similar to the human sequence, showing differences at 34 positions only. However, the cloned BoAChE sequence differs from the published amino acid sequence of AChE isolated from fetal bovine serum (FBS) by: (1) 13 amino acids, 12 of which are conserved between BoAChE and human AChE, and (2) the presence of four rather than five potential N-glycosylation sites. The full coding sequence of the mature BoAChE T-subunit was expressed in human embryonal kidney 293 cells (HEK-293). The catalytic properties of recombinant BoAChE and its reactivity towards various inhibitors were similar to those of the native bovine enzyme. Soluble recombinant BoAChE is composed of monomers, dimers and tetramers, yet in contrast to FBS-AChE, tetramer formation is not efficient. Comparative SDS/PAGE analysis reveals that all four potential N-glycosylation sites identified by DNA sequencing appear to be utilized, and that recombinant BoAChE comigrates with FBS-AChE. A major difference between the recombinant enzyme and the native enzyme was observed when clearance from circulation was examined. The HEK-293-derived enzyme was cleared from the circulation at a much faster rate than FBS-AChE. This difference in behaviour, together with previous studies on the effect of post-translation modification on human AChE clearance [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959-967] suggests that cell-dependent glycosylation plays a key role in AChE circulatory residence.
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