Tracking Modified Vaccinia Virus Ankara in the Chicken Embryo: In Vivo Tropism and Pathogenesis of Egg Infections

The Modified Vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus serving as a promising vector vaccine platform to develop vaccines against infectious diseases. In contrast to the well-established replication deficiency and safety of MVA in mammals, much less is known about MVA infection in avian hosts. Here, we used a recombinant MVA expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters to study in vivo tropism, distribution, and pathogenesis of MVA infections in embryonated chicken eggs. The chorioallantoic membrane (CAM) of embryonated chicken eggs was inoculated with recombinant MVA, MVA or phosphate-buffered saline. The infection was analyzed by fluorescence microscopy, histology, immunohistochemistry, and virus titration of embryonic tissues. After infection of the CAM, MVA spread to internal and external embryonic tissues with the liver as a major target organ. Macrophages and hematopoietic cells were identified as primary target cells of MVA infection and may be involved in virus spread. Increasing doses of MVA did not result in increased lesion severity or embryonic death. Despite MVA generalization to embryonic tissues, the CAM seems to be the major site of MVA replication. The absence of considerable organ lesions and MVA-associated mortality highlights an excellent safety profile of MVA in chicken hosts

Newly designed poxviral promoters to improve immunogenicity and efficacy of MVA-NP candidate vaccine against lethal influenza virus infection in mice

Influenza, a respiratory disease mainly caused by influenza A and B, viruses of the Orthomyxoviridae, is still a burden on our society's health and economic system. Influenza A viruses (IAV) circulate in mammalian and avian populations, causing seasonal outbreaks with high numbers of cases. Due to the high variability in seasonal IAV triggered by antigenic drift, annual vaccination is necessary, highlighting the need for a more broadly protective vaccine against IAV. The safety tested Modified Vaccinia virus Ankara (MVA) is licensed as a third-generation vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against different pathogens. Here, we generated and characterized recombinant MVA candidate vaccines that deliver the highly conserved internal nucleoprotein (NP) of IAV under the transcriptional control of five newly designed chimeric poxviral promoters to further increase the immunogenic properties of the recombinant viruses (MVA-NP). Infections of avian cell cultures with the recombinant MVA-NPs demonstrated efficient synthesis of the IAV-NP which was expressed under the control of the five new promoters. Prime-boost or single shot immunizations in C57BL/6 mice readily induced circulating serum antibodies' binding to recombinant IAV-NP and the robust activation of IAV-NP-specific CD8+ T cell responses. Moreover, the MVA-NP candidate vaccines protected C57BL/6 mice against lethal respiratory infection with mouse-adapted IAV (A/Puerto Rico/8/1934/H1N1). Thus, further studies are warranted to evaluate the immunogenicity and efficacy of these recombinant MVA-NP vaccines in other IAV challenge models in more detail

Cartilage and Soft Tissue Imaging Using X-rays

International audienceObjectivesThis study evaluates high-resolution tomographic x-ray phase-contrast imaging in whole human knee joints for the depiction of soft tissue with emphasis on hyaline cartilage. The method is compared with conventional computed tomography (CT), synchrotron radiation absorption-based CT, and magnetic resonance imaging (MRI).Material and MethodsAfter approval of the institutional review board, 2 cadaveric human knees were examined at an synchrotron institution using a monochromatic x-ray beam of 60 keV, a detector with a 90-mm2 field of view, and a pixel size of 46 × 46 μm2. Images of phase-contrast imaging CT were reconstructed with the filtered back projection algorithm and the equally sloped tomography method. Image quality and tissue contrast were evaluated and compared in all modalities and with histology.ResultsPhase-contrast imaging provides visualization of altered cartilage regions invisible in absorption CT with simultaneous high detail of the underlying bony abnormalities. The delineation of surface changes is similar to 3-T MRI using cartilage-dedicated sequences. Phase-contrast imaging CT presents soft tissue contrast surpassing that of conventional CT with a clear discrimination of ligamentous, muscular, neural, and vascular structures. In addition, phase-contrast imaging images show cartilage and meniscal calcifications that are not perceptible on conventional CT or on MRI.ConclusionsPhase-contrast imaging CT may facilitate a more complete evaluation of the human knee joint by providing concurrent comprehensive information about cartilage, the underlying subchondral bone, and their changes in osteoarthritic conditions

Translation of atherosclerotic plaque phase-contrast CT imaging from synchrotron radiation to a conventional lab-based X-ray source.

OBJECTIVES: Phase-contrast imaging is a novel X-ray based technique that provides enhanced soft tissue contrast. The aim of this study was to evaluate the feasibility of visualizing human carotid arteries by grating-based phase-contrast tomography (PC-CT) at two different experimental set-ups: (i) applying synchrotron radiation and (ii) using a conventional X-ray tube. MATERIALS AND METHODS: Five ex-vivo carotid artery specimens were examined with PC-CT either at the European Synchrotron Radiation Facility using a monochromatic X-ray beam (2 specimens; 23 keV; pixel size 5.4 µm), or at a laboratory set-up on a conventional X-ray tube (3 specimens; 35-40 kVp; 70 mA; pixel size 100 µm). Tomographic images were reconstructed and compared to histopathology. Two independent readers determined vessel dimensions and one reader determined signal-to-noise ratios (SNR) between PC-CT and absorption images. RESULTS: In total, 51 sections were included in the analysis. Images from both set-ups provided sufficient contrast to differentiate individual vessel layers. All PCI-based measurements strongly predicted but significantly overestimated lumen, intima and vessel wall area for both the synchrotron and the laboratory-based measurements as compared with histology (all p0.53 per mm(2), 95%-CI: 0.35 to 0.70). Although synchrotron-based images were characterized by higher SNRs than laboratory-based images; both PC-CT set-ups had superior SNRs compared to corresponding conventional absorption-based images (p0.98 and >0.84 for synchrotron and for laboratory-based measurements; respectively). CONCLUSION: Experimental PC-CT of carotid specimens is feasible with both synchrotron and conventional X-ray sources, producing high-resolution images suitable for vessel characterization and atherosclerosis research

Emphysema diagnosis using X-ray dark-field imaging at a laser-driven compact synchrotron light source

In early stages of various pulmonary diseases, such as emphysema and fibrosis, the change in X-ray attenuation is not detectable with absorption-based radiography. To monitor the morphological changes that the alveoli network undergoes in the progression of these diseases, we propose using the dark-field signal, which is related to small-angle scattering in the sample. Combined with the absorption-based image, the dark-field signal enables better discrimination between healthy and emphysematous lung tissue in a mouse model. All measurements have been performed at 36 keV using a monochromatic laser-driven miniature synchrotron X-ray source (Compact Light Source). In this paper we present grating-based dark-field images of emphysematous vs. healthy lung tissue, where the strong dependence of the dark-field signal on mean alveolar size leads to improved diagnosis of emphysema in lung radiographs

Comparison of PC-CT images and absorption CT images obtained with a conventional X-ray tube; and corresponding histology sections.

<p>(A) Axial reconstructed PC-CT slice from the conventional X-ray tube, (B) axial reconstructed CT slice from the conventional X-ray tube, (C) and (D) corresponding histology sections. This atherosclerotic lesion shows a large lipid-rich necrotic core and a relatively thin fibrous cap. The arrows point to the fibrous cap (FC), the lipid-rich necrotic core (NC) and an area of calcification (CA). Several other calcifications are seen in this specimen (PC-CT = Phase Contrast Computed Tomography; CT = Computed Tomography, Length of the scale bar = 2 mm).</p

Three-dimensional renderings of the phase contrast tomographic data sets.

<p>(A) Near-normal carotid artery imaged at the synchrotron beamline ID19, ESRF, France. Due to the high resolution of the images and the excellent soft tissue contrast, several vasa vasorum can be identified without the use of contrast dye. (B) Atherosclerotic lesion imaged at the laboratory (Images were rendered using VGStudioMax 2.1, Volume Graphics, Heidelberg, Germany)..</p