Advanced Development of the rF1V and rBV A/B Vaccines: Progress and Challenges

The development of vaccines for microorganisms and bacterial toxins with the potential to be used as biowarfare and bioterrorism agents is an important component of the US biodefense program. DVC is developing two vaccines, one against inhalational exposure to botulinum neurotoxins A1 and B1 and a second for Yersinia pestis, with the ultimate goal of licensure by the FDA under the Animal Rule. Progress has been made in all technical areas, including manufacturing, nonclinical, and clinical development and testing of the vaccines, and in assay development. The current status of development of these vaccines, and remaining challenges are described in this chapter

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V

Characterization of a Bacillus anthracis spore coat-surface protein that influences coat-surface morphology

Bacterial spores are encased in a multilayered proteinaceous shell, called the coat. In many Bacillus spp., the coat protects against environmental assault and facilitates germination. In Bacillus anthracis , the spore is the etiological agent of anthrax, and the functions of the coat likely contribute to virulence. Here, we characterize a B. anthracis spore protein, called CotΒ, which is encoded only in the genomes of the Bacillus cereus group. We found that CotΒ is synthesized specifically during sporulation and is assembled onto the spore coat surface. Our analysis of a cotΒ null mutant in the Sterne strain reveals that CotΒ has a role in determining coat-surface morphology but does not detectably affect germination. In the fully virulent Ames strain, a cotΒ null mutation has no effect on virulence in a murine model of B. anthracis infection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72138/1/j.1574-6968.2008.01380.x.pd

Allelic Variation on Murine Chromosome 11 Modifies Host Inflammatory Responses and Resistance to Bacillus anthracis

Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT), as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6) background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36–74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT

Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment

Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream

Contributions of chaperone/usher systems to cell binding, biofilm formation and Yersinia pestis virulence

Yersinia pestis genome sequencing projects have revealed six intact uncharacterized chaperone/ usher systems with the potential to play roles in plague pathogenesis. We cloned each locus and expressed them in the Deltafim Escherichia coli strain AAEC185 to test the assembled Y. pestis surface structures for various activities. Expression of each chaperone/usher locus gave rise to specific novel fibrillar structures on the surface of E. coli. One locus, y0561-0563, was able to mediate attachment to human epithelial cells (HEp-2) and human macrophages (THP-1) but not mouse macrophages (RAW264.7), while several loci were able to facilitate E. coli biofilm formation. When each chaperone/usher locus was deleted in Y. pestis, only deletion of the previously described pH 6 antigen (Psa) chaperone/usher system resulted in decreased adhesion and biofilm formation. Quantitative RT-PCR (qRT-PCR) revealed low expression levels for each novel chaperone/usher system in vitro as well as in mouse tissues following intravenous infection. However, a Y. pestis mutant in the chaperone/usher locus y1858-1862 was attenuated for virulence in mice via the intravenous route of infection, suggesting that expression of this locus is, at some stage, sufficient to affect the outcome of a plague infection. qRT-PCR experiments also indicated that expression of the chaperone/usher-dependent capsule locus, caf1, was influenced by oxygen availability and that the well-described chaperone/usher-dependent pilus, Psa, was strongly induced in minimal medium even at 28 degrees C rather than 37 degrees C, a temperature previously believed to be required for Psa expression. These data indicate several potential roles for the novel chaperone/usher systems of Y. pestis in pathogenesis and infection-related functions such as cell adhesion and biofilm formation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91950/1/2011 Microbiology - Contributions of chaperone usher systems to cell binding biofilm formation and Yersinia pestis virulence.pd

Anthrax Toxins in Context of Bacillus anthracis Spores and Spore Germination

The interaction of anthrax toxin or toxin components with B. anthracis spores has been demonstrated. Germinating spores can produce significant amounts of toxin components very soon after the initiation of germination. In this review, we will summarize the work performed that has led to our understanding of toxin and spore interactions and discuss the complexities associated with these interaction

Modified Caspase-3 Assay Indicates Correlation of Caspase-3 Activity with Immunity of Nonhuman Primates to Yersinia pestis Infection▿

Defined candidate human vaccines for treating infection by Yersinia pestis, the agent of plague, have been developed. To facilitate evaluation of the vaccines' efficacy, the in vitro caspase-3 assay for cytotoxicity-neutralizing activity was modified and reevaluated. Immune serum-mediated caspase-3 neutralizing activity correlated with protection against infection in a nonhuman primate vaccine model of plague immunity

Roles of Macrophages and Neutrophils in the Early Host Response to Bacillus anthracis Spores in a Mouse Model of Infection

The development of new approaches to combat anthrax requires that the pathogenesis and host response to Bacillus anthracis spores be better understood. We investigated the roles that macrophages and neutrophils play in the progression of infection by B. anthracis in a mouse model. Mice were treated with a macrophage depletion agent (liposome-encapsulated clodronate) or with a neutrophil depletion agent (cyclophosphamide or the rat anti-mouse granulocyte monoclonal antibody RB6-8C5), and the animals were then infected intraperitoneally or by aerosol challenge with fully virulent, ungerminated B. anthracis strain Ames spores. The macrophage-depleted mice were significantly more susceptible to the ensuing infection than the saline-pretreated mice, whereas the differences observed between the neutropenic mice and the saline-pretreated controls were generally not significant. We also found that augmenting peritoneal neutrophil populations before spore challenge did not increase resistance of the mice to infection. In addition, the bacterial load in macrophage-depleted mice was significantly greater and appeared significantly sooner than that observed with the saline-pretreated mice. However, the bacterial load in the neutropenic mice was comparable to that of the saline-pretreated mice. These data suggest that, in our model, neutrophils play a relatively minor role in the early host response to spores, whereas macrophages play a more dominant role in early host defenses against infection by B. anthracis spores