Live-attenuated CHIKV vaccine with rearranged genome replicates in vitro and induces immune response in mice.

Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. CHIKV is considered a priority pathogen by CEPI and WHO. Despite recent approval of a live-attenuated CHIKV vaccine, development of additional vaccines is warranted due to the worldwide outbreaks of CHIKV. Previously, we developed immunization DNA (iDNA) plasmid capable of launching live-attenuated CHIKV vaccine in vivo. Here we report the use of CHIKV iDNA plasmid to prepare a novel, live-attenuated CHIKV vaccine V5040 with rearranged RNA genome. In V5040, genomic RNA was rearranged to encode capsid gene downstream from the glycoprotein genes. Attenuated mutations derived from experimental CHIKV 181/25 vaccine were also engineered into E2 gene of V5040. The DNA copy of rearranged CHIKV genomic RNA with attenuated mutations was cloned into iDNA plasmid pMG5040 downstream from the CMV promoter. After transfection in vitro, pMG5040 launched replication of V5040 virus with rearranged genome and attenuating E2 mutations. Furthermore, V5040 virus was evaluated in experimental murine models for general safety and immunogenicity. Vaccination with V5040 virus subcutaneously resulted in elicitation of CHIKV-specific, virus-neutralizing antibodies. The results warrant further evaluation of V5040 virus with rearranged genome as a novel live-attenuated vaccine for CHIKV.

Development and evaluation of murine lung-specific disease models for Pseudomonas aeruginosa applicable to therapeutic testing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing a wide range of disease manifestations, including severe bacterial pneumonia. Recently, clinics have reported a rise in nosocomial infections with multidrug resistant (MDR) species, including MDR strains of P. aeruginosa. In order to quickly evaluate the efficacy of new therapeutics for MDR infections, highly reproducible and validated animal models need to be developed for pre-clinical testing. Here, we describe the characterization of two murine models to study MDR P. aeruginosa respiratory disease. We evaluated and compared these models using a non-invasive intratracheal instillation method and established the 50% lethal dose, course of infection, biometric parameters of disease and degree of pneumonia development for each model. Further, we tested meropenem as a proof-of-concept therapeutic and report efficacy data that suggests that the leukopenic model could serve a robust pre-clinical model to test novel therapeutics.

Molecular imaging reveals a progressive pulmonary inflammation in lower airways in ferrets infected with 2009 H1N1 pandemic influenza virus.

Molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. Herein we used [(18)F]-2-deoxy-2-fluoro-D-glucose ([(18)F]-FDG) as a radiotracer for PET imaging coupled with CT (FDG-PET/CT) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, H1N1 (H1N1pdm). The thoracic regions of mock- and H1N1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (DPI). On 1 DPI, FDG-PET/CT imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [(18)F]-FDG uptake (maximum standardized uptake values (SUVMax), 4.7-7.0). By days 2 and 3, consolidation (CT) and inflammation ([(18)F]-FDG) appeared in the left caudal lobe. By 6 DPI, CT images showed extensive areas of patchy ground-glass opacities (GGO) and consolidations with the largest lesions having high SUVMax (6.0-7.6). Viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 DPI, but not on day 7, respectively. In conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on CT with corresponding [(18)F]-FDG uptake. Strong positive correlations were measured between SUVMax and bronchiolitis-related pathologic scoring (Spearman's ρ = 0.75). Importantly, the extensive areas of patchy GGO and consolidation seen on CT in the ferret model at 6 DPI are similar to that reported for human H1N1pdm infections. In summary, these first molecular imaging studies of lower respiratory infection with H1N1pdm show that FDG-PET can give insight into the spatiotemporal progression of the inflammation in real-time.

Ferret thoracic anatomy by 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) positron emission tomography/computed tomography (18F-FDG PET/CT) imaging.

The domestic ferret (Mustela putorius furo) has been a long-standing animal model used in the evaluation and treatment of human diseases. Molecular imaging techniques such as 2-deoxy-2-((18)F)fluoro-D-glucose ((18)F-FDG) positron emission tomography (PET) would be an invaluable method of tracking disease in vivo, but this technique has not been reported in the literature. Thus, the aim of this study was to establish baseline imaging characteristics of PET/computed tomography (CT) with (18)F-FDG in the ferret model. Twelve healthy female ferrets were anesthetized and underwent combined PET/CT scanning. After the images were fused, volumes of interest (VOIs) were generated in the liver, heart, thymus, and bilateral lung fields. For each VOI, standardized uptake values (SUVs) were calculated. Additional comparisons were made between radiotracer uptake periods (60, 90, and >90 minutes), intravenous and intraperitoneal injections of (18)F-FDG, and respiratory gated and ungated acquisitions. Pulmonary structures and the surrounding thoracic and upper abdominal anatomy were readily identified on the CT scans of all ferrets and were successfully fused with PET. VOIs were created in various tissues with the following SUV calculations: heart (maximum standardized uptake value [SUV(Max)] 8.60, mean standardized uptake value [SUV(Mean)] 5.42), thymus (SUV(Max) 3.86, SUV(Mean) 2.59), liver (SUV(Max) 1.37, SUV(Mean) 0.99), right lung (SUV(Max) 0.92, SUV(Mean) 0.56), and left lung (SUV(Max) 0.88, SUV(Mean) 0.51). Sixty- to 90-minute uptake periods were sufficient to separate tissues based on background SUV activity. No gross differences in image quality were seen between intraperitoneal and intravenous injections of (18)F-FDG. Respiratory gating also did not have a significant impact on image quality of lung parenchyma. The authors concluded that (18)F-FDG PET and CT imaging can be performed successfully in normal healthy ferrets with the parameters identified in this study. They obtained similar imaging features and uptake measurements with and without respiratory gating as well as with intraperitoneal and intravenous (18)F-FDG injections. (18)F-FDG PET and CT can be a valuable resource for the in vivo tracking of disease progression in future studies that employ the ferret model.