Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.

Melioidosis is an endemic disease caused by the bacterium Burkholderia pseudomallei. Concerns exist regarding B. pseudomallei use as a potential bio-threat agent causing persistent infections and typically manifesting as severe pneumonia capable of causing fatal bacteremia. Development of suitable therapeutics against melioidosis is complicated due to high degree of genetic and phenotypic variability among B. pseudomallei isolates and lack of data establishing commonly accepted strains for comparative studies. Further, the impact of strain variation on virulence, disease presentation, and mortality is not well understood. Therefore, this study evaluate and compare the virulence and disease progression of B. pseudomallei strains K96243 and HBPUB10303a, following aerosol challenge in a standardized BALB/c mouse model of infection. The natural history analysis of disease progression monitored conditions such as weight, body temperature, appearance, activity, bacteremia, organ and tissue colonization (pathological and histological analysis) and immunological responses. This study provides a detailed, direct comparison of infection with different B. pseudomallei strains and set up the basis for a standardized model useful to test different medical countermeasures against Burkholderia species. Further, this protocol serves as a guideline to standardize other bacterial aerosol models of infection or to define biomarkers of infectious processes caused by other intracellular pathogens.

We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (⌬caf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the ⌬caf mutant was devoid of a capsule. The growth rate of the ⌬caf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutant's growth dropped slightly during the late phase at 37°C. The ⌬caf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 g/ml of purified F1 antigen or 1 ؋ 10 5 to 5 ؋ 10 5 CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 ؋ 10 8 CFU/ml of the ⌬caf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. It produces edema toxin (EdTx), a powerful adenylate cyclase that increases cyclic AMP (cAMP) levels in host cells. Because other cAMP-increasing agents inhibit key macrophage (M⌽) functions, such as phagocytosis, it was hypothesized that EdTx would exhibit similar suppressive activities. Our previous GeneChip data showed that EdTx downregulated M⌽ genes involved in actin cytoskeleton remodeling, including protein kinase A (PKA). To further examine the role of EdTx during anthrax pathogenesis, we explored the hypothesis that EdTx treatment leads to deregulation of the cAMP-dependent PKA system, resulting in impaired cytoskeletal functions essential for M⌽ activity. Our data revealed that EdTx significantly suppressed human M⌽ phagocytosis of Ames spores. Cytoskeletal changes, such as decreased cell spreading and lowered F-actin content, were also observed for toxin-treated M⌽s. Further, EdTx altered the protein levels and activity of PKA and exchange protein activated by cAMP (Epac), a recently identified cAMP-binding molecule. By using PKA-and Epac-selective cAMP analogs, we confirmed the involvement of both pathways in the inhibition of M⌽ functions elicited by EdTx-generated cAMP. These results suggested that EdTx weakened the host immune response by increasing cAMP levels, which then signaled via PKA and Epac to cripple M⌽ phagocytosis and interfered with cytoskeletal remodeling.

The respiratory epithelium is a dynamic interface between the outside environment and the interior of the host (20, 56). Protection against respiratory infection is provided by the physical barrier formed by alveolar epithelial cells (AECs), which also are vital for maintaining lung homeostasis. AECs are abundant in number and line the pulmonary airways and alveoli. Alveolar type I (ATI) cells are the epithelial component of the thin air-blood barrier and comprise Ͼ95% of the alveolar surface area (57). Alveolar type II (ATII) cells cover approximately 4% of the mammalian alveolar surface but constitute 15% of all lung cells (8,9,20,35,57).