Newly Designed Poxviral Promoters to Improve Immunogenicity and Efficacy of MVA-NP Candidate Vaccines 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.

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.

Distribution and absence of generalized lesions in mice following single dose intramuscular inoculation of the vaccine candidate MVA-MERS-S.

Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient virus serving as vaccine against infectious diseases. Here, we assessed the in vivo distribution of a recombinant MVA candidate vaccine against the Middle Eastern Respiratory Syndrome (MVA-MERS-S) in mice. Intramuscularly inoculated mice were necropsied at different time points and examined by histology, immunohistochemistry and real-time PCR. We detected inflammation and myonecrosis at the parenteral site and hyperplasia of the draining lymph nodes. MVA-MERS-S did not result in detectable lesions in tissues peripheral to the parenteral site and draining lymph nodes. Real-time PCR analysis of >240 tissue samples detected MVA-DNA predominantly at the injection site and in the draining lymph nodes, and suggested continuous clearance of the candidate vaccine during the observation period. Levels of parenteral site inflammation and hyperplasia of draining lymph nodes were considered in line with immunological responses to vaccine inoculation.

Quantitative Assessment of Degenerative Cartilage and Subchondral Bony Lesions in a Preserved Cadaveric Knee: Propagation-Based Phase-Contrast CT Versus Conventional MRI and CT.

OBJECTIVE: The aim of this study was to quantitatively assess hyaline cartilage and subchondral bone conditions in a fully preserved cadaveric human knee joint using high-resolution x-ray propagation-based phase-contrast imaging (PBI) CT and to compare the performance of the new technique with conventional CT and MRI. MATERIALS AND METHODS: A cadaveric human knee was examined using an x-ray beam of 60 keV, a detector with a 90-mm2 FOV, and a pixel size of 46 × 46 µm2. PBI CT images were reconstructed with both the filtered back projection algorithm and the equally sloped tomography method. Conventional 3-T MRI and CT were also performed. Measurements of cartilage thickness, cartilage lesions, International Cartilage Repair Society scoring, and detection of subchondral bone changes were evaluated. Visual inspection of the specimen akin to arthroscopy was conducted and served as a standard of reference for lesion detection. RESULTS: Loss of cartilage height was visible on PBI CT and MRI. Quantification of cartilage thickness showed a strong correlation between the two modalities. Cartilage lesions appeared darker than the adjacent cartilage on PBI CT. PBI CT showed similar agreement to MRI for depicting cartilage substance defects or lesions compared with the visual inspection. The assessment of subchondral bone cysts showed moderate to strong agreement between PBI CT and CT. CONCLUSION: In contrast to the standard clinical methods of MRI and CT, PBI CT is able to simultaneously depict cartilage and bony changes at high resolution. Though still an experimental technique, PBI CT is a promising high-resolution imaging method to evaluate comprehensive changes of osteoarthritic disease in a clinical setting.

Cartilage and soft tissue imaging using X-rays: propagation-based phase-contrast computed tomography of the human knee in comparison with clinical imaging techniques and histology.

OBJECTIVES: This 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 METHODS: After 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-mm field of view, and a pixel size of 46 × 46 µm. 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. RESULTS: Phase-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. CONCLUSIONS: Phase-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.

Phase-contrast CT: qualitative and quantitative evaluation of atherosclerotic carotid artery plaque.

PURPOSE: To evaluate the potential of phase-contrast computed tomography (CT) for atherosclerotic plaque imaging in human carotid arteries in an experimental ex vivo study. MATERIALS AND METHODS: The study was approved by the institutional review board, and informed consent was obtained from the patients' relatives. Seven postmortem human carotid artery specimens were imaged at a laboratory setup by using a conventional x-ray tube and grating interferometer. After histologic processing, phase-contrast imaging and histopathologic data were matched. Characteristics of the necrotic core (NC) covered by a fibrous cap (FC), intraplaque hemorrhage (IPH), and calcifications (CAs) were established, and sensitivity, specificity, and accuracy of phase-contrast CT for plaque detection and the potential for accurate quantification were assessed. The Cohen κ and Pearson correlation coefficient R were used to determine the agreement between phase-contrast imaging and histopathologic findings for plaque characterization and correlation of quantitative plaque measurements, respectively. A difference with a P value of less than .05 was considered significant. RESULTS: Characteristic criteria were found in all analyzed plaque components. Applying these criteria, phase-contrast CT had a good sensitivity for the detection of the FC and NC, IPH, and CAs (all, >80%) and excellent specificity and accuracy (all, >90%), with good interreader agreement (κ ≥ 0.72, P < .0001). There were excellent correlations for quantitative measurements of FC, NC, and CAs between phase-contrast imaging and histopathologic findings (R ≥ 0.92). Interreader reproducibility was excellent, with an intraclass correlation coefficient of 0.98 or higher for all measurements. CONCLUSION: The results of this study indicate that ex vivo phase-contrast CT can help identify and quantify atherosclerotic plaque components, with excellent correlation to histopathologic findings. Although not yet applicable in vivo, phase-contrast CT may become a valuable tool to monitor atherosclerotic disease process noninvasively.

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 p<0.001 with slope >0.53 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 (p<0.001). Inter-reader reproducibility was excellent (ICCs >0.98 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.

Grating-based X-ray phase-contrast tomography of atherosclerotic plaque at high photon energies.

BACKGROUND: Tissue characterization of atherosclerosis by absorption-based imaging methods is limited due to low soft-tissue contrast. Grating-based phase-contrast computed tomography (PC-CT) may become an alternative for plaque assessment if the phase signal can be retrieved at clinically applicable photon energies. The aims of this feasibility study were (i) to characterize arterial vessels at low and high photon energies, (ii) to extract qualitative features and (iii) quantitative phase-contrast Hounsfield units (HU-phase) of plaque components at 53 keV using histopathology as gold standard. MATERIALS AND METHODS: Five human carotid artery specimens underwent grating-based PC-CT using synchrotron radiation of either 23 keV or 53 keV and histological work-up. Specimens without advanced atherosclerosis were used to extract signal criteria of vessel layers. Diseased specimens were screened for important plaque components including fibrous tissue (FT), lipid (LIP), necrotic core (NEC), intraplaque hemorrhage (IPH), inflammatory cell infiltration (INF) and calcifications (CA). Qualitative features as well as quantitative HU-phase were analyzed. RESULTS: Thirty-three regions in 6 corresponding PC-CT scans and histology sections were identified. Healthy samples had the same signal characteristics at 23 keV and 53 keV with bright tunica intima and adventitia and dark media. Plaque components showed differences in signal intensity and texture at 53 keV. Quantitative analysis demonstrated the highest HU-phase of soft plaque in dense FT. Less organized LIP, NEC and INF were associated with lower HU-phase values. The highest HU-phase were measured in CA. CONCLUSION: PC-CT of atherosclerosis is feasible at high, clinically relevant photon energies and provides detailed information about plaque structure including features of high risk vulnerable plaques.

Diffusion-tensor imaging of human articular cartilage specimens with early signs of cartilage damage.

PURPOSE: To assess the use of diffusion-tensor (DT) imaging of articular cartilage to detect and grade early cartilage damage in human specimens with early signs of cartilage damage. MATERIALS AND METHODS: This study was approved by the institutional review board. Forty-three cartilage-on-bone samples drilled from 21 human patellae were examined with 17.6-T magnetic resonance (MR) imaging and a diffusion-weighted spin-echo sequence (spatial resolution, 50 × 100 × 800 µm). Subsequently, samples underwent histologic analysis with safranin O staining. Cartilage damage on safranin O histologic slides was quantified with Osteoarthritis Research Society International (OARSI) grades; grades ranged from 0 (healthy) to 6 (bone remodeling). Maps of longitudinal diffusivity (λ(l)), transverse diffusivity (λ(t)), mean diffusivity (MD), and fractional anisotropy (FA) were calculated. Cartilage was segmented, and region of interest (ROI) analysis was performed and compared with histologic findings. Significant differences in MR parameters between the OARSI groups were assessed with the Tukey test. The value of DT imaging in the diagnosis and grading of cartilage damage was assessed with logistic regression analysis. RESULTS: Samples had OARSI grades of 0 (n = 14), 1 (n = 11), 2 (n = 12), 3 (n = 4), and 4 (n = 2). Samples with an OARSI grade greater than 0 had significantly increased λ(l), λ(t), and MD (7%-25% increase) in the superficial cartilage growing deeper into cartilage with increasing OARSI grade. Samples with an OARSI grade greater than 0 showed significantly decreased FA in the deep cartilage (-25% to -35% decrease), suggesting that changes in the collagen architecture may occur early in cartilage degradation. DTI showed excellent performance in the detection of cartilage damage (accuracy, 0.95; 41 of 43 samples) and good performance in the grading of cartilage damage (accuracy, 0.74; 32 of 43 samples). CONCLUSION: DT imaging of articular cartilage can enable physicians to detect and grade early cartilage damage.

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.

Ultra-high field diffusion tensor imaging of articular cartilage correlated with histology and scanning electron microscopy.

OBJECT: To investigate the relationship of the different diffusion tensor imaging (DTI) parameters (ADC, FA, and first eigenvector (EV)) to the constituents (proteoglycans and collagen), the zonal arrangement of the collagen network, and mechanical loading of articular cartilage. MATERIAL AND METHODS: DTI of eight cartilage-on-bone samples of healthy human patellar cartilage was performed at 17.6 T. Three samples were additionally imaged under indentation loading. After DTI, samples underwent biomechanical testing, safranin-O staining for semiquantitative proteoglycan estimation, and scanning electron microscopy (SEM) for depicting collagen architecture. RESULTS: From the articular surface to the bone-cartilage interface, ADC continuously decreased and FA increased. Cartilage zonal heights calculated from EVs strongly correlated with SEM-derived zonal heights (P < 0.01, r (2)=0.87). Compression reduced ADC in the superficial 30% of cartilage and increased FA in the superficial 5% of cartilage. Reorientation of the EVs indicative of collagen fiber reorientation under the indenter was observed. No significant correlation was found between ADC, FA, and compressive stiffness. CONCLUSIONS: Correlating ADC and FA with proteoglycan and collagen content suggests that diffusion is dominated by different depth-dependent mechanisms within cartilage. Knowledge of the spatial distribution of the DTI parameters and their variation contributes to form a database for future analysis of defective cartilage.

Change of diffusion tensor imaging parameters in articular cartilage with progressive proteoglycan extraction.

OBJECTIVE: To investigate changes of diffusion tensor imaging (DTI) parameters (mean apparent diffusion coefficient [ADC], fractional anisotropy [FA], and first eigenvector) with increasing proteoglycan (PG) extraction of articular cartilage. MATERIAL AND METHODS: Twelve cylindrical cartilage-on-bone samples were drilled from 4 human patellae (3 per patella). Each sample was divided into 2 pieces. One piece underwent histologic examination to assess the PG content of the native sample by safranin-O staining and its collagen architecture by polarized light microscopy. The other underwent magnetic resonance imaging (MRI) at 17.6 T for DTI measurement. After MRI, 2 of the 3 samples from each patella were immersed in a dilute trypsin solution (0.1 mg/mL), whereas the third sample was kept as a negative control in physiological saline. After incubation (6, 48, 72, and 96 hours), the samples were reimaged, stained for PG content and for the collagen orientation. Maps of ADC, FA, and the orientation of the first eigenvector as well as histology were available for each sample before and after incubation. RESULTS: PG loss led to increased ADC and reduced safranin-O staining from the articular surface to the bone-cartilage interface. A significant correlation (r(2) = 0.86, P < 0.01) was observed between the change in bulk ADC and PG loss. Regional analysis from the articular surface to the tide mark demonstrated depth dependent significant correlations of ADC and PG loss. FA and first eigenvector as well as polarized light microscopy showed only small changes in the order of magnitude of measurement errors, not correlating with PG loss. CONCLUSION: Mean diffusivity evidence by the ADC is linearly correlated to progressive PG extraction in articular cartilage. FA and the first eigenvector seem to be specific to the collagen architecture of cartilage. DTI has the potential to become a valuable biomarker for the workup of cartilage degeneration in osteoarthritis, since evaluation of the PG content and collagen architectural properties of cartilage can be performed with a single, non­contrast-enhanced proton-based MRI measurement.

Characterization of osteoarthritic and normal human patella cartilage by computed tomography X-ray phase-contrast imaging: a feasibility study.

OBJECTIVES: Early research in phase-contrast imaging indicates that substantial higher soft-tissue contrast resolution can be obtained compared with conventional absorption radiography. In the present feasibility study, we used the phase contrast analyzer-based technique in tomographic mode to investigate whether structural cartilage matrix properties can be depicted in an ex vivo set-up and whether high resolution CT-phase contrast imaging may enable differentiation of osteoarthritic and intact cartilage matrixes. MATERIAL AND METHODS: Four postmortem osteochondral cylinders (7 mm diameter, 2 osteoarthritic, 2 healthy control samples from 4 human patellae) underwent tomographic phase-contrast analyzer-based imaging at high resolution (voxel size: 8(3) micron3) at 26 keV (European Synchrotron Radiation Facility, Grenoble, France). From the acquired data volumes, sets of reconstructed sagittal slices were selected at 0.5 mm increments from osteoarthritic and control specimens. Two independent, blinded observers assessed structural characteristics (cartilage thickness, topographic chondrocyte distribution homogeneity, zonal height, and surface damage) and differences between the 2 groups were determined. RESULTS: Phase contrast analyzer-based CT showed excellent depiction of the complete volume and of the 3D architecture of the cartilage in all samples. A distinct zonal pattern in the cartilage matrix could consistently be visualized. The osteoarthritic samples showed significantly lower chondrocyte distribution homogeneity (0% vs. 76% homogeneous, P < 0.001), less chondrocyte alignment (0% vs. 59% fully aligned, P < 0.001), lower height of tangential, transitional, and radial zones (all P < 0.001) and a higher prevalence of superficial cartilage damage (84% vs. 10%, P < 0.001). CONCLUSIONS: This first proof-of-concept study demonstrates that high resolution phase contrast CT visualizes structural details in relatively thick ex vivo cartilage samples. Our results suggest that the technique permits differentiation of osteoarthritic and healthy cartilage by enabling assessment of histologic characteristics of cartilage structures.