Search

The aerosol characteristics of Rift Valley fever virus (RVFV) were evaluated to achieve reproducible infection of experimental animals with aerosolized RVFV suitable for animal efficacy studies. Spray factor (SF), the ratio between the concentrations of the aerosolized agent to the agent in the aerosol generator, is used to compare performance differences between aerosol exposures. SF indicates the efficiency of the aerosolization process; a higher SF means a lower nebulizer concentration is needed to achieve a desired inhaled dose. Relative humidity levels as well as the duration of the exposure and choice of exposure chamber all impacted RVFV SF. Differences were also noted between actual and predicted minute volumes for different species of nonhuman primates. While NHP from Old World species (Macaca fascicularis, M. mulatta, Chlorocebus aethiops) generally had a lower actual minute volume than predicted, the actual minute volume for marmosets (Callithrix jacchus) was higher than predicted (150% for marmosets compared with an average of 35% for all other species examined). All of these factors (relative humidity, chamber, duration, and minute volume) impact the ability to reliably and reproducibly deliver a specific dose of aerosolized RVFV. The implications of these findings for future pivotal efficacy studies are discussed.

Using a comprehensive set of discovery and optimization tools, antibodies were produced with the ability to neutralize SARS coronavirus (SARS-CoV) infection in Vero E6 cells and in animal models. These anti-SARS antibodies were discovered using a novel DNA display method, which can identify new antibodies within days. Once neutralizing antibodies were identified, a comprehensive and effective means of converting the mouse sequences to human frameworks was accomplished using HuFR™ (human framework reassembly) technology. The best variant (61G4) from this screen showed a 3.5–4-fold improvement in neutralization of SARS-CoV infection in vitro. Finally, using a complete site-saturation mutagenesis methodology focused on the CDR (complementarity determining regions), a single point mutation (51E7) was identified that improved the 80% plaque reduction neutralization of the virus by greater than 8-fold. These discovery and evolution strategies can be applied to any emerging pathogen or toxin where a causative agent is known.

Host responses to Venezuelan equine encephalitis viruses (VEEV) were studied in cynomolgus macaques after aerosol exposure to the epizootic virus. Changes in global gene expression were assessed for the brain, lungs, and spleen. In the brain, major histocompatibility complex (MHC) class I transcripts were induced, while the expression of S100b, a factor associated with brain injury, was inhibited, as was expression of the encephalitogenic gene MOG. Cytokine-mediated signals were affected by infection, including those involving IFN-mediated antiviral activity (IRF-7, OAS, and Mx transcripts), and the increased transcription of caspases. Induction of a few immunologically relevant genes (e.g. IFITM1 and STAT1) was common to all tested tissues. Herein, both tissue-specific and nontissue specific transcriptional changes in response to VEEV are described, including induction of IFN-regulated transcripts and cytokine-induced apoptotic factors, in addition to cellular factors in the brain that may be descriptive of the health status of the brain during the infectious process. Altogether, this work provides novel information on common and tissue-specific host responses against VEEV in a nonhuman primate model of aerosol exposure.

Cynomolgus macaques exposed to an aerosol containing a virulent strain of eastern equine encephalitis (EEE) virus developed neurological signs indicating encephalitis that corresponded with the onset of fever and an elevated heart rate. Viremia was either transient or undetectable even in animals that succumbed to the illness. The onset of illness was dose dependent, but once a febrile response was observed, macaques were moribund within 36 h. Simultaneously, a prominent leukocytosis was seen; 1 day before being moribund, macaques had a white blood cell count >20,000 cells/ microL. The leukocytes were predominantly granulocytes. Increases in serum levels of blood urea nitrogen, sodium, and alkaline phosphatase were also seen. The rapid onset and severity of neurological signs mirror what has been reported for human cases of disease caused by EEE.

Because Venezuelan equine encephalitis viruses (VEEVs) are infectious by aerosol, they are considered to be a biological-weapons threat. Nonhuman-primate models are needed to evaluate the efficacy of candidate vaccines. In the present study, cynomolgus macaques, after aerosol exposure to either VEEV-IE or VEEV-IIIA, developed fever, viremia, and lymphopenia; the severity of the fever response, viremia, and lymphopenia correlated with the inhaled dose of VEEV. Of the 10 macaques in our study, 7 developed clinical signs indicative of encephalitis, including loss of balance and hypothermia. In the macaque, the enzootic strains used are infectious by aerosol and lead to disease, including clinical encephalitis.

Tularemia is a severe, zoonotic disease caused by a gram-negative bacterium, Francisella tularensis. We have previously shown that rabbits are a good model of human pneumonic tularemia when exposed to aerosols containing a virulent, type A strain, SCHU S4. We further demonstrated that the live vaccine strain (LVS), an attenuated type B strain, extended time to death when given by scarification. Oral or aerosol vaccination has been previously shown in humans to offer superior protection to parenteral vaccination against respiratory tularemia challenge. Both oral and aerosol vaccination with LVS were well tolerated in the rabbit with only minimal fever and no weight loss after inoculation. Plasma antibody titers against F. tularensis were higher in rabbits that were vaccinated by either oral or aerosol routes compared to scarification. Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4. LVS given by scarification extended time to death compared to mock-vaccinated controls. One orally vaccinated rabbit did survive aerosol challenge, however, only aerosol vaccination extended time to death significantly compared to scarification. These results further demonstrate the utility of the rabbit model of pneumonic tularemia in replicating what has been reported in humans and macaques as well as demonstrating the utility of vaccination by oral and respiratory routes against an aerosol tularemia challenge.