A Bacteriophage T4 Nanoparticle-Based Dual Vaccine against Anthrax and Plague.

Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, are two of the deadliest pathogenic bacteria that have been used as biological warfare agents. Although Biothrax is a licensed vaccine against anthrax, no Food and Drug Administration-approved vaccine exists for plague. Here, we report the development of a dual anthrax-plague nanoparticle vaccine employing bacteriophage (phage) T4 as a platform. Using an in vitro assembly system, the 120- by 86-nm heads (capsids) of phage T4 were arrayed with anthrax and plague antigens fused to the small outer capsid protein Soc (9 kDa). The antigens included the anthrax protective antigen (PA) (83 kDa) and the mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system from Y. pestis (F1mutV) (56 kDa). These viral nanoparticles elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria. Unlike the traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. Therefore, phage T4 is a unique nanoparticle platform to formulate multivalent vaccines against high-risk pathogens for national preparedness against potential bioterror attacks and emerging infections.IMPORTANCE Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance.

Small molecule inhibitors of Bacillus anthracis protective antigen proteolytic activation and oligomerization.

Protective antigen (PA), lethal factor, and edema factor, the protein toxins of Bacillus anthracis , are among its most important virulence factors and play a key role in infection. We performed a virtual ligand screen of a library of 10000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA association in a FRET-based oligomerization assay, and two of those protected cells from intoxication at concentrations of 1-10 µM. Exploration of the protective mechanism by Western blot showed decreased SDS-resistant PA oligomer on cells and, surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA's cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins.

Anthrax edema toxin has cAMP-mediated stimulatory effects and high-dose lethal toxin has depressant effects in an isolated perfused rat heart model.

While anthrax edema toxin produces pronounced tachycardia and lethal toxin depresses left ventricular (LV) ejection fraction in in vivo models, whether these changes reflect direct cardiac effects as opposed to indirect ones related to preload or afterload alterations is unclear. In the present study, the effects of edema toxin and lethal toxin were investigated in a constant pressure isolated perfused rat heart model. Compared with control hearts, edema toxin at doses comparable to or less than a dose that produced an 80% lethality rate (LD(80)) in vivo in rats (200, 100, and 50 ng/ml) produced rapid increases in heart rate (HR), coronary flow (CF), LV developed pressure (LVDP), dP/dt(max), and rate-pressure product (RPP) that were most pronounced and persisted with the lowest dose (P ≤ 0.003). Edema toxin (50 ng/ml) increased effluent and myocardial cAMP levels (P ≤ 0.002). Compared with dobutamine, edema toxin produced similar myocardial changes, but these occurred more slowly and persisted longer. Increases in HR, CF, and cAMP with edema toxin were inhibited by a monoclonal antibody blocking toxin uptake and by adefovir, which inhibits the toxin's intracellular adenyl cyclase activity (P ≤ 0.05). Lethal toxin at an LD(80) dose (50 ng/ml) had no significant effect on heart function but a much higher dose (500 ng/ml) reduced all parameters (P ≤ 0.05). In conclusion, edema toxin produced cAMP-mediated myocardial chronotropic, inotropic, and vasodilatory effects. Vasodilation systemically with edema toxin could contribute to shock during anthrax while masking potential inotropic effects. Although lethal toxin produced myocardial depression, this only occurred at high doses, and its relevance to in vivo findings is unclear.

Inhibition of tumor angiogenesis by the matrix metalloproteinase-activated anthrax lethal toxin in an orthotopic model of anaplastic thyroid carcinoma.

Patients with anaplastic thyroid carcinoma (ATC) typically succumb to their disease months after diagnosis despite aggressive therapy. A large percentage of ATCs have been shown to harbor the V600E B-Raf point mutation, leading to the constitutive activation of the mitogen-activated protein kinase pathway. ATC invasion, metastasis, and angiogenesis are in part dependent on the gelatinase class of matrix metalloproteinases (MMP). The explicit targeting of these two tumor markers may provide a novel therapeutic strategy for the treatment of ATC. The MMP-activated anthrax lethal toxin (LeTx), a novel recombinant protein toxin combination, shows potent mitogen-activated protein kinase pathway inhibition in gelatinase-expressing V600E B-Raf tumor cells in vitro. However, preliminary in vivo studies showed that the MMP-activated LeTx also exhibited dramatic antitumor activity against xenografts that did not show significant antiproliferative responses to the LeTx in vitro. Here, we show that the MMP-activated LeTx inhibits orthotopic ATC xenograft progression in both toxin-sensitive and toxin-resistant ATC cells via reduced endothelial cell recruitment and subsequent tumor vascularization. This in turn translates to an improved long-term survival that is comparable with that produced by the multikinase inhibitor sorafenib. Our results also indicate that therapy with the MMP-activated LeTx is extremely effective against advanced tumors with well-established vascular networks. Taken together, these results suggest that the MMP-activated LeTx-mediated endothelial cell targeting is the primary in vivo antitumor mechanism of this novel toxin. Therefore, the MMP-activated LeTx could be used not only in the clinical management of V600E B-Raf ATC but potentially in any solid tumor.

An antibody-based microarray assay for small RNA detection.

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.

Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop.

The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the 83 kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts. The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts. Maximum levels of expression were observed in mature leaves under continuous illumination. Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF). Crude leaf extracts contained up to 25 microg functional PA/ml. With an average yield of 172 mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field.