Different pathologies but equal levels of responsiveness to the recombinant F1 and V antigen vaccine and ciprofloxacin in a murine model of plague caused by small- and large-particle aerosols.

Presently there is a significant effort to develop and evaluate vaccines and antibiotics against the potential bioterrorism agent Yersinia pestis. The animal models used to test these countermeasures involve the deposition of small particles within the lung. However, deliberate aerosol release of Y. pestis will generate both small and large inhalable particles. We report in this study that the pathogenesis patterns of plague infections caused by the deposition of 1- and 12-microm-particle aerosols of Y. pestis in the lower and upper respiratory tracts (URTs) of mice are different. The median lethal dose for 12-mum particles was 4.9-fold greater than that for 1-microm particles. The 12-microm-particle infection resulted in the degradation of the nasal mucosa and nasal-associated lymphoid tissue (NALT) plus cervical lymphadenopathy prior to bacteremic dissemination. Lung involvement was limited to secondary pneumonia. In contrast, the 1-microm-particle infection resulted in primary pneumonia; in 40% of mice, the involvement of NALT and cervical lymphadenopathy were observed, indicating entry via both URT lymphoid tissues and lungs. Despite bacterial deposition in the gastrointestinal tract, the involvement of Peyer's patches was not observed in either infection. Although there were major differences in pathogenesis, the recombinant F1 and V antigen vaccine and ciprofloxacin protected against plague infections caused by small- and large-particle aerosols.

Characterization and deposition of respirable large- and small-particle bioaerosols.

The deposition patterns of large-particle microbiological aerosols within the respiratory tract are not well characterized. A novel system (the flow-focusing aerosol generator [FFAG]) which enables the generation of large (>10-microm) aerosol particles containing microorganisms under laboratory conditions was characterized to permit determination of deposition profiles within the murine respiratory tract. Unlike other systems for generating large aerosol particles, the FFAG is compatible with microbiological containment and the inhalational challenge of animals. By use of entrapped Escherichia coli cells, Bacillus atrophaeus spores, or FluoSphere beads, the properties of aerosols generated by the FFAG were compared with the properties of aerosols generated using the commonly available Collison nebulizer, which preferentially generates small (1- to 3-microm) aerosol particles. More entrapped particulates (15.9- to 19.2-fold) were incorporated into 9- to 17-microm particles generated by the FFAG than by the Collison nebulizer. The 1- to 3-microm particles generated by the Collison nebulizer were more likely to contain a particulate than those generated by the FFAG. E. coli cells aerosolized using the FFAG survived better than those aerosolized using the Collison nebulizer. Aerosols generated by the Collison nebulizer and the FFAG preferentially deposited in the lungs and nasal passages of the murine respiratory tract, respectively. However, significant deposition of material also occurred in the gastrointestinal tract after inhalation of both the small (89.7%)- and large (61.5%)-particle aerosols. The aerosols generated by the Collison nebulizer and the FFAG differ with respect to mass distribution, distribution of the entrapped particulates, bacterial survival, and deposition within the murine respiratory tract.

The identification and evaluation of ATP binding cassette systems in the intracellular bacterium Francisella tularensis.

Francisella tularensis is a facultative intracellular bacterium responsible for the disease tularemia. Analysis of the fully sequenced genome of the virulent F. tularensis strain SCHU S4 has led to the identification of twenty ATP binding cassette (ABC) systems, of which five appear to be non-functional. The fifteen complete systems comprise three importers, five exporters, four systems involved in non-transport processes, and three systems of unknown or ill-defined function. The number and classification of the ABC systems in F. tularensis is similar to that observed in other intracellular bacteria, indicating that some of these systems may be important for the intracellular lifestyle of these organisms. Among the ABC systems identified in the genome are systems that may be involved in the virulence of F. tularensis SCHU S4. Six ABC system proteins were evaluated as candidate vaccine antigens against tularemia, although none provided significant protection against F. tularensis. However, a greater understanding of these systems may lead to the development of countermeasures against F. tularensis.

Antibiotic-free plasmid stabilization by operator-repressor titration for vaccine delivery by using live Salmonella enterica Serovar typhimurium.

Live, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of Salmonella enterica serovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed lacO sequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyer's patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the Yersinia pestis F1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with Salmonella expressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.