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Mortality from plague is high if not treated with the proper antibiotics within 18-24 hours after onset of symptoms. The process of antibiotic susceptibility determination of isolated from blood samples may extend from 4 to more than 7 days, since the growth is very slow. To accelerate this process, we developed an enrichment protocol as well as a non-standard yet reliable method for rapid antibiotic susceptibility analysis of from blood cultures using flow cytometry technology. This rapid method is applicable to blood cultures containing low levels of .

The performance of a single-tube nested-PCR (STNPCR) technique was evaluated for plague diagnosis in comparison to conventional (one step) and two step nested PCR (NPCR). Assays were carried out with primers targeting the gene that encodes the F1 antigen. For STNPCR inner primers were immobilized onto the inside of the microtube caps and after the first amplification they were eluted by inversion of the tube. This procedure avoids opening the tube, reducing the risks of false-positive results by crosscontamination. The immobilized primers are stable for several months at –20°C, thus, the tubes can be prepared beforehand and stored until use. STNPCR was more sensitive than conventional PCR, and less sensitive than NPCR. This drawback is compensated by a lower risk of cross-contamination.

Microbial pathogens have developed various stratagems for modulating and/or circumventing the host’s innate and adaptive immunity. Hence, certain virulence factors can be viewed as potential therapeutic agents for human immunopathological diseases. This is the case for virulence plasmid-encoded proteins from pathogenic Yersiniae that inhibit the host’s inflammatory response by interfering with various cellular signaling pathways.

, and , utilize a plasmid encoded type III secretion system (T3SS) to promote infection by delivering Yersinia outer proteins (Yops) into the cytosol of mammalian cells. This T3SS is absolutely required for virulence, which makes T3SS an attractive target in the development of novel therapeutics for treatment of plague and other infections. In this study, a new method for high throughput screening (HTS) of small molecules for the ability to inhibit type III secretion (T3S) in has been developed. In comparison with screening assays employed by others, this method is very simple and rapid, and thus well suited for examining very large compound sets. Using this method, we screened a diverse collection of libraries at the US National Screening Laboratory. The initial examination of 70,966 compounds and mixtures from 13 libraries resulted in 431 primary hits. Strong positive indications of inhibition were observed at a rate of 0.01%, while moderate and weak but potentially meaningful signals were observed at rates of 0.056% and 0.54% respectively. Further characterizations were conducted on selected primary hits in . Of the eight compounds examined in secondary assays, four show good promise as leads for structure activity relationship studies. They are a diverse group, each having chemical scaffolds not only distinct from one another, but also distinct from previously described candidate T3S inhibitors.

() - one of the world’s most deadly human pathogens - is the gram-negative bacterium that causes pneumonic plague. Virulent antibiotic-resistant strains exist and Cold War scientists devised means to effectively aerosolize . These facts raise grave concern that will be exploited as a bioweapon. To counter that possibility, it is essential that we develop a safe and effective pneumonic plague vaccine. Recent studies suggest that the leading vaccine candidate, which primarily stimulates antibody-mediated humoral immunity, may not suffice. T cell-dependent cellular immunity comprises a second means by which vaccines prime long-lived protection against virulent bacterial pathogens. However, a plasmid carried by virulent encodes factors that dampen inflammation and debilitate phagocytes, thereby compromising cellular defense mechanisms. As such, plague vaccine researchers have devoted relatively little attention to cellular immunity. Here we review our recent work demonstrating that the passive transfer of primed T cells can suffice to protect mice against lethal intranasal infection, a model of pneumonic plague. We also demonstrate that key elements of cellular immunity even play critical roles during antibody-mediated defense against plague. We conclude that next-generation plague vaccines should strive to prime both cellular and humoral immunity.

The use of live recombinant attenuated vaccine (RASV) encoding proteins is a promising new approach for the vaccination against We have tested the efficacy of 2 proteins, Psn and a portion of LcrV in protecting mice against virulent challenge. To remove the immunosuppressive properties of LcrV protein, the gene, without the TLR2 receptor sequence, was cloned into a β-lactamase secretion vector. Immunizations were performed with RSAV expressing LcrV or Psn. Challenge with a virulent strain was performed 4 weeks after the last immunization. Our results show that the truncated LcrV protein delivered by RASV is sufficient to afford a full protective immune response in a mouse model of bubonic plague and the Psn protein afforded partial protection in a non-optimized system. This finding should facilitate the design and development of a new generation of vaccines against .

Current subunit vaccines provide partial protection against pneumonic plague if the infecting strain is encapsulated (F1+). Here we describe YadC, a novel outer membrane protein that provides partial protection against a F1– strain. Swiss-Webster mice were immunized subcutaneously with glutathione S-transferase (GST) or His6-tagged (HT) purified fusion proteins (GST-YadC137-409 or HT-LcrV) or buffer emulsified with Alhydrogel. Intravenous challenge with 1 x 104 F1– CO99-3015 revealed no protection for those mice immunized with GST-Alhydrogel alone, full protection for HT-LcrVimmunized mice, and partial protection for GST-YadC137-409 -immunized mice. Similarly, C57BL/6 mice were immunized with GST-YadC137-409, HT-LcrV, or GST all with Alhydrogel adjuvant. After intranasal challenge with 3 x 103 F1– CO99-3015, 87% of GSTYadC137- 409-immunized mice survived pneumonic plague. This is compared to the GST control group (0 surviving mice) and the LcrV-immunized group where 50% survived the challenge. This protection was correlated with a predominantly IgG1 response in LcrV-immunized mice and an IgG1/IgG3 antibody response in YadC-immunized mice. Additionally, we report the cytokine response from HT-LcrV- and GST-YadC137-409-stimulated peripherally derived macrophages. YadC-stimulated cells demonstrated a predominant pro-inflammatory cytokine production. This mixed Th1/Th2 response suggests that YadC’s protection may involve a different adaptive immune response than the LcrV protein that currently is part of plague vaccines.

Plague, an infectious disease that reached catastrophic proportions during three pandemics, continues to be a legitimate public health concern worldwide. Although antibiotic therapy for the causative agent is available, pharmaceutical supply limitations, multi-drug resistance from natural selection as well as malicious bioengineering are a reality. Consequently, plague vaccinology is a priority for biodefense research. Development of a multi-subunit vaccine with Fraction 1 and LcrV as protective antigens seems to be receiving the most attention. However, LcrV has been shown to cause immune suppression and mutants lacking F1 expression are thought to be fully virulent in nature and in animal experiments. The LcrV variant, rV10, retains the well documented protective antigenic properties of LcrV but with diminished inhibitory effects on the immune system. More research is required to examine the molecular mechanisms of vaccine protection afforded by surface protein antigens and to decipher the host mechanisms responsible for vaccine success.