SUMMARY A murine low-dose (LD) aerosol model is commonly used to test tuberculosis vaccines. Doses of 50-400 CFU (24-hour lung CFU) infect 100% of exposed mice. The LD model measures progression from infection to disease based on organ CFU at defined time points. To mimic natural exposure, we exposed mice to an ultra-low dose (ULD) aerosol. We estimated the presented dose by sampling the aerosol. Female C57BL/6 mice were exposed to Mycobacterium tuberculosis H37Rv aerosol at 1.0, 1.1, 1.6, 5.4, and 11 CFU presented dose, infecting 27%, 36%, 36%, 100%, and 95% of mice, respectively. These data are compatible with a stochastic infection event (Poisson distribution, weighted R2= 0.97) or with a dose-response relationship (sigmoid distribution, weighted R2= 0.97). Based on the later assumption, the ID50 was 1.6 CFU presented dose (95% confidence interval, 1.2 to 2.1). We compared organ CFU after ULD and LD aerosols (5.4 vs. 395 CFU presented dose). Lung burden was 30-fold lower in the ULD model at 4 weeks (3.4 vs. 4.8 logs, p<0.001) and 18 weeks (≤3.6 vs. 5.0 logs, p=0.01). Mice exposed to ULD aerosols as compared to LD aerosols had greater within-group CFU variability. Exposure to ULD aerosols leads to infection in a subset of mice, and to persistently low organ CFU. The ULD aerosol model may resemble human pulmonary tuberculosis more closely than the standard LD model, and may be used to identify host or bacterial factors that modulate the initial infection event.
Introduction Multiple factors influence the viability of aerosolized bacteria. The delivery of aerosols is affected by chamber conditions (humidity, temperature, and pressure) and bioaerosol characteristics (particle number, particle size distribution, and viable aerosol concentration). Measurement of viable aerosol concentration and particle size is essential to optimize viability and lung delivery. The Madison chamber is widely used to expose small animals to infectious aerosols. Methods A multiplex sampling port was added to the Madison chamber to measure the chamber conditions and bioaerosol characteristics. Aerosols of three pathogens (Bacillus anthracis, Yersinia pestis, and Mycobacterium tuberculosis) were generated under constant conditions and their bioaerosol characteristics were analyzed. Airborne microbes were captured using an impinger or BioSampler. The particle size distribution of airborne microbes was determined using an aerodynamic particle sizer (APS). Viable aerosol concentration, spray factor (viable aerosol concentration/inoculum concentration), and dose presented to the mouse were calculated. Dose retention efficiency and viable aerosol retention rate were calculated from the sampler titers to determine the efficiency of microbe retention in lungs of mice. Results B. anthracis, Y. pestis, and M. tuberculosis aerosols were sampled through the port. The count mean aerodynamic sizes were 0.98, 0.77, and 0.78 μm with geometric standard deviations of 1.60, 1.90, and 2.37, and viable aerosol concentrations in the chamber were 211, 57, and 1 colony-forming unit (CFU)/mL, respectively. Based on the aerosol concentrations, the doses presented to mice for the three pathogens were 2.5e5, 2.2e4 and 464 CFU. Discussion Using the multiplex sampling port we determined whether the animals were challenged with an optimum bioaerosol based on dose presented and respirable particle size.
The adaptive immune response to Francisella tularensis is dependent on the route of inoculation. Intradermal inoculation with the F. tularensis live vaccine strain (LVS) results in a robust Th1 response in the lungs, whereas intranasal inoculation produces fewer Th1 cells and instead many Th17 cells. Interestingly, bacterial loads in the lungs are similar early after inoculation by these two routes. We hypothesize that the adaptive immune response is influenced by local events in the lungs, such as the type of cells that are first infected with Francisella. Using fluorescence-activated cell sorting, we identified alveolar macrophages as the first cell type infected in the lungs of mice intranasally inoculated with F. novicida U112, LVS, or F. tularensis Schu S4. Following bacterial dissemination from the skin to the lung, interstitial macrophages or neutrophils are infected. Overall, we identified the early interactions between Francisella and the host following two different routes of inoculation.
The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ⌬leuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freezethaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ⌬leuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10 24 -fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10 68 -fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ⌬leuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. The immense global burden of human immunodeficiency virus (HIV) infection necessitates the development of an efficacious vaccine. There is increasing interest in the use of live recombinant bacterial vectors as HIV vaccines due to the inherent advantages of utilizing a replicating antigen delivery system that is itself an effective adjuvant (1, 2). Previous studies have examined the use of live Gram-positive and Gram-negative bacterial vectors, including recombinant Salmonella, Listeria, Streptococcus, and Escherichia coli, for heterologous expression of HIV antigens, with varying success (3-8).Mycobacterium bovis BCG is the most widely administered vaccine in the world (9). Its extensively documented safety in immunocompetent individuals, relatively low production cost, and well-established infrastructure for vaccine administration make it an ideal candidate for use as an anti-HIV vaccine vehicle (10-12). In addition to the logistical advantages of using BCG, mycobacterial antigen delivery systems possess inherent adjuvant properties which activate innate immunity (13,14). Mycobacteria such as BCG ...
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