Human infections with highly pathogenic avian influenza A (H5N1) virus are frequently fatal but the mechanisms of disease remain ill-defined. H5N1 infection is associated with intense production of proinflammatory cytokines, but whether this cytokine storm is the main cause of fatality or is a consequence of extensive virus replication that itself drives disease remains controversial. Conventional intratracheal inoculation of a liquid suspension of H5N1 influenza virus in nonhuman primates likely results in efficient clearance of virus within the upper respiratory tract and rarely produces severe disease. We reasoned that small particle aerosols of virus would penetrate the lower respiratory tract and blanket alveoli where target cells reside. We show that inhalation of aerosolized H5N1 influenza virus in cynomolgus macaques results in fulminant pneumonia that rapidly progresses to acute respiratory distress syndrome with a fatal outcome reminiscent of human disease. Molecular imaging revealed intense lung inflammation coincident with massive increases in proinflammatory proteins and interferon-α in distal airways. Aerosolized H5N1 exposure decimated alveolar macrophages, which were widely infected and caused marked influx of interstitial macrophages and neutrophils. Extensive infection of alveolar epithelial cells caused apoptosis and leakage of albumin into airways, reflecting loss of epithelial barrier function. These data establish inhalation of aerosolized virus as a critical source of exposure for fatal human infection and reveal that direct viral effects in alveoli mediate H5N1 disease. This new nonhuman primate model will advance vaccine and therapeutic approaches to prevent and treat human disease caused by highly pathogenic avian influenza viruses.

Experimental infection of animals with aerosols containing pathogenic agents is essential for an understanding of the natural history and pathogenesis of infectious disease as well as evaluation of potential treatments. We evaluated whether the Aeroneb nebulizer, a vibrating mesh nebulizer, would serve as an alternative to the Collison nebulizer, the “gold standard” for generating infectious bioaerosols. While the Collison possesses desirable properties that have contributed to its longevity in infectious disease aerobiology, concerns have lingered about the liquid volume and concentration of the infectious agent required to cause disease and the damage that jet nebulization causes to the agent. Fluorescein salt was added to the nebulizer contents to assess pathogen loss during aerosolization. Relative to fluorescein salt, loss of influenza virus during aerosolization was worse with the Collison than with the Aeroneb. Four other viruses also had superior aerosol performance with the Aeroneb. The Aeroneb did not improve the aerosol performance for a vegetative bacterium, Francisella tularensis. Environmental parameters collected during the aerosol challenges indicated that the Aeroneb generated a higher relative humidity in exposure chambers while not affecting other environmental parameters. The aerosol mass median aerodynamic diameter (MMAD) was generally larger and more disperse for aerosols generated by the Aeroneb than what is seen with the Collison, but ≥80% of particles were within the range that would reach the lower respiratory tract and alveolar regions. The improved aerosol performance and generated particle size range suggest that for viral pathogens, the Aeroneb is a suitable alternative to the Collison three-jet nebulizer for use in experimental infection of animals. IMPORTANCE Respiratory infection by pathogens via aerosol remains a major concern for both natural disease transmission as well as intentional release of biological weapons. Critical to understanding the disease course and pathogenesis of inhaled pathogens are studies in animal models conducted under tightly controlled experimental settings, including the inhaled dose. The route of administration, particle size, and dose can affect disease progression and outcome. Damage to or loss of pathogens during aerosolization could increase the dose required to cause disease and could stimulate innate immune responses, altering outcome. Aerosol generators that reduce pathogen loss would be ideal. This study compares two aerosol generators to determine which is superior for animal studies. Aerosol research methods and equipment need to be well characterized to optimize the development of animal models for respiratory pathogens, including bioterrorism agents. This information will be critical for pivotal efficacy studies in animals to evaluate potential vaccines or treatments against these agents.

Tularemia, also known as rabbit fever, is a severe zoonotic disease in humans caused by the gram-negative bacterium Francisella tularensis (Ft). While there have been a number of attempts to develop a vaccine for Ft, few candidates have advanced beyond experiments in inbred mice. We report here that a prime-boost strategy with aerosol delivery of recombinant live attenuated candidate Ft S4ΔaroD offers significant protection (83% survival) in an outbred animal model, New Zealand White rabbits, against aerosol challenge with 248 cfu (11 LD50) of virulent type A Ft SCHU S4. Surviving rabbits given two doses of the attenuated strains by aerosol did not exhibit substantial post-challenge fevers, changes in erythrocyte sedimentation rate or in complete blood counts. At a higher challenge dose (3,186 cfu; 139 LD50), protection was still good with 66% of S4ΔaroD-vaccinated rabbits surviving while 50% of S4ΔguaBA vaccinated rabbits also survived challenge. Pre-challenge plasma IgG titers against Ft SCHU S4 corresponded with survival time after challenge. Western blot analysis found that plasma antibody shifted from predominantly targeting Ft O-antigen after the prime vaccination to other antigens after the boost. These results demonstrate the superior protection conferred by a live attenuated derivative of virulent F. tularensis, particularly when given in an aerosol prime-boost regimen.

17Experimental infection of animals via inhalation containing pathogenic agents is essential to 18 understanding the natural history and pathogenesis of infectious disease as well as evaluation of 19 potential medical countermeasures. We evaluated whether the Aeroneb, a vibrating mesh 20 nebulizer, would serve as an alternative to the Collison, the 'gold standard' for generating 21 infectious bioaerosols. While the Collison possesses desirable properties that have contributed to 22 its longevity in infectious disease aerobiology, concerns have lingered about the volume and 23 concentration of agent required to cause disease and the damage that jet nebulization causes to 24 the agent. For viruses, the ratio of aerosol concentration to nebulizer concentration (spray factor, 25 SF), the Aeroneb was superior to the Collison for four different viruses in a nonhuman primate 26 head-only exposure chamber. Aerosol concentration of influenza was higher relative to 27 fluorescein for the Aeroneb compared to the Collison, suggesting that the Aeroneb was less 28 harsh to viral pathogens than the Collison when generating aerosols. The Aeroneb did not 29 improve the aerosol SF for a vegetative bacterium, Francisella tularensis. Environmental 30 parameters collected during the aerosols indicated that the Aeroneb generated a higher relative 31 humidity in exposure chambers while not affecting other environmental parameters. Aerosol 32 mass median aerodynamic diameter was generally larger and more disperse for aerosols 33 generated by the Aeroneb than what is seen with the Collison but ≥80% were within the range 34 that would reach the lower respiratory tract and alveolar regions. These data suggest that for viral 35 pathogens, the Aeroneb is a suitable alternative to the Collison 3-jet nebulizer. 36 Importance 37 The threat of aerosolization is often not the natural method of transmission. While selection of 38 an appropriate animal model is vital for these types of experiments, other confounding factors 39 can be controlled through a thorough understanding of experimental design and the effects that 40 different parameters can have on disease outcome. Route of administration, particle size, and 41 dose are all factors which can affect disease progression and need to be controlled. Aerosol 42 research methods and equipment need to be well characterized to optimize the development of 43 animal models for bioterrorism agents.44 45 65responses that protect the host. These effects could raise the dose required to cause disease, 66 thereby requiring large quantities of pathogens grown to high titers for aerosol experiments. 67While the process of aerosolization will always place mechanical stress on infectious agents, 68 aerosol generators that are 'gentler' than the Collison would be desirable. 69The Aerogen Solo (a.k.a Aeroneb) is a single-use nebulizer employed in clinical settings 70 for the delivery of aerosolized medication. The Aeroneb utilizes a palladium mesh perforated 71 with conical shaped holes that act as a micropump...

Inhalation of Francisella tularensis causes pneumonic tularemia in humans, a severe disease with a 30 to 60% mortality rate. The reproducible delivery of aerosolized virulent bacteria in relevant animal models is essential for evaluating medical countermeasures. Here we developed optimized protocols for infecting New Zealand White (NZW) rabbits with aerosols containing F. tularensis. We evaluated the relative humidity, aerosol exposure technique, and bacterial culture conditions to optimize the spray factor (SF), a central metric of aerosolization. This optimization reduced both inter-and intraday variability and was applicable to multiple isolates of F. tularensis. Further improvements in the accuracy and precision of the inhaled pathogen dose were achieved through enhanced correlation of the bacterial culture optical density and the number of CFU. Plethysmograph data collected during exposures found that respiratory function varied considerably between rabbits, was not a function of weight, and did not improve with acclimation to the system. Live vaccine strain (LVS)-vaccinated rabbits were challenged via aerosol with human-virulent F. tularensis SCHU S4 that had been cultivated in either Mueller-Hinton broth (MHB) or brain heart infusion (BHI) broth. LVS-vaccinated animals challenged with SCHU S4 that had been cultivated in MHB experienced short febrile periods (median, 3.2 days), limited weight loss (Ͻ5%), and longer median survival times (ϳ18 days) that were significantly different from those for unvaccinated controls. In contrast, LVS-vaccinated rabbits challenged with SCHU S4 that had been cultivated in BHI experienced longer febrile periods (median, 5.5 days) and greater weight loss (Ͼ10%) than the unvaccinated controls and median survival times that were not significantly different from those for the unvaccinated controls. These studies highlight the importance of careful characterization and optimization of protocols for aerosol challenge with pathogenic agents.