A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.

Synthesis and Preclinical Evaluation of PSMA-Targeted 111In-Radioconjugates Containing a Mitochondria-Tropic Triphenylphosphonium Carrier.

Nuclear DNA is the canonical target for biological damage induced by Auger electrons (AE) in the context of targeted radionuclide therapy (TRT) of cancer, but other subcellular components might also be relevant for this purpose, such as the energized mitochondria of tumor cells. Having this in mind, we have synthesized novel DOTA-based chelators carrying a prostate-specific membrane antigen (PSMA) inhibitor and a triphenyl phosphonium (TPP) group that were used to obtain dual-targeted 111In-radioconjugates ([111In]In-TPP-DOTAGA-PSMA and [111In]In-TPP-DOTAGA-G3-PSMA), aiming to promote a selective uptake of an AE-emitter radiometal (111In) by PSMA+ prostate cancer (PCa) cells and an enhanced accumulation in the mitochondria. These dual-targeted 111In-radiocomplexes are highly stable under physiological conditions and in cell culture media. The complexes showed relatively similar binding affinities toward the PSMA compared to the reference tracer [111In]In-PSMA-617, in line with their high cellular uptake and internalization in PSMA+ PCa cells. The complexes compromised cell survival in a dose-dependent manner and in the case of [111In]In-TPP-DOTAGA-G3-PSMA to a higher extent than observed for the single-targeted congener [111In]In-PSMA-617. µSPECT imaging studies in PSMA+ PCa xenografts showed that the TPP pharmacophore did not interfere with the excellent in vivo tumor uptake of the "golden standard" [111In]In-PSMA-617, although it led to a higher kidney retention. Such kidney retention does not necessarily compromise their usefulness as radiotherapeutics due to the short tissue range of the Auger/conversion electrons emitted by 111In. Overall, our results provide valuable insights into the potential use of mitochondrial targeting by PSMA-based radiocomplexes for efficient use of AE-emitting radionuclides in TRT, giving impetus to extend the studies to other AE-emitting trivalent radiometals (e.g., 161Tb or 165Er) and to further optimize the designed dual-targeting constructs.

Innovating in a bioimaging core through instrument development.

Developing devices and instrumentation in a bioimaging core facility is an important part of the innovation mandate inherent in the core facility model but is a complex area due to the required skills and investments, and the impossibility of a universally applicable model. Here, we seek to define technological innovation in microscopy and situate it within the wider core facility innovation portfolio, highlighting how strategic development can accelerate access to innovative imaging modalities and increase service range, and thus maintain the cutting edge needed for sustainability. We consider technology development from the perspective of core facility staff and their stakeholders as well as their research environment and aim to present a practical guide to the 'Why, When, and How' of developing and integrating innovative technology in the core facility portfolio. Core facilities need to innovate to stay up to date. However, how to carry out the innovation is not very obvious. One area of innovation in imaging core facilities is the building of optical setups. However, the creation of optical setups requires specific skill sets, time, and investments. Consequently, the topic of whether a core facility should develop optical devices is discussed as controversial. Here, we provide resources that should help get into this topic, and we discuss different options when and how it makes sense to build optical devices in core facilities. We discuss various aspects, including consequences for staff and the relation of the core to the institute, and also broaden the scope toward other areas of innovation.

Staying on track - Keeping things running in a high-end scientific imaging core facility.

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation.

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Challenges and advances in optical 3D mesoscale imaging.

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence.

This review discusses the state of the art of an emerging field called mesoscale imaging. Mesoscale imaging refers to the trend towards imaging ever-larger samples that exceed the classic microscopy domain and is also referred to as 'mesoscopic imaging'. In optical imaging, this refers to objects between the microscopic and macroscopic scale that are imaged with subcellular resolution; in practice, this implies the imaging of objects from millimetre up to cm size with µm or nm resolution. As such, the mesoscopy field spans the boundary between classic 'biological' imaging and preclinical 'biomedical' imaging, typically utilising lower magnification objective lenses with a bigger field of view. We discuss the types of samples currently imaged with examples, and highlight how this type of imaging fills the gap between microscopic and macroscopic imaging, allowing further insight into the organisation of tissues in an organism. We also discuss the challenges of imaging such large samples, from sample handling to labelling and optical phenomena that stand in the way of quantitative imaging. Finally, we put the current state of the art into context within the neighbouring fields and outline future developments, such as the use of 'phantom' test samples and artificial intelligence for image analysis that will underpin the quality of mesoscale imaging.

Collaborating by courier, imaging by mail.

Core facilities offer visiting scientists access to equipment and expertise to generate and analyze data. For some projects, it might however be more efficient to collaborate remotely by sending in samples.

A workflow for streamlined acquisition and correlation of serial regions of interest in array tomography.

BACKGROUND: Array tomography (AT) is a high-resolution imaging method to resolve fine details at the organelle level and has the advantage that it can provide 3D volumes to show the tissue context. AT can be carried out in a correlative way, combing light and electron microscopy (LM, EM) techniques. However, the correlation between modalities can be a challenge and delineating specific regions of interest in consecutive sections can be time-consuming. Integrated light and electron microscopes (iLEMs) offer the possibility to provide well-correlated images and may pose an ideal solution for correlative AT. Here, we report a workflow to automate navigation between regions of interest. RESULTS: We use a targeted approach that allows imaging specific tissue features, like organelles, cell processes, and nuclei at different scales to enable fast, directly correlated in situ AT using an integrated light and electron microscope (iLEM-AT). Our workflow is based on the detection of section boundaries on an initial transmitted light acquisition that serves as a reference space to compensate for changes in shape between sections, and we apply a stepwise refinement of localizations as the magnification increases from LM to EM. With minimal user interaction, this enables autonomous and speedy acquisition of regions containing cells and cellular organelles of interest correlated across different magnifications for LM and EM modalities, providing a more efficient way to obtain 3D images. We provide a proof of concept of our approach and the developed software tools using both Golgi neuronal impregnation staining and fluorescently labeled protein condensates in cells. CONCLUSIONS: Our method facilitates tracing and reconstructing cellular structures over multiple sections, is targeted at high resolution ILEMs, and can be integrated into existing devices, both commercial and custom-built systems.

Clinical validation of the novel HDAC6 radiotracer [18F]EKZ-001 in the human brain.

PURPOSE: Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that modulates intracellular transport and protein quality control. Inhibition of HDAC6 deacetylase activity has shown beneficial effects in disease models, including Alzheimer's disease and amyotrophic lateral sclerosis. This first-in-human positron emission tomography (PET) study evaluated the brain binding of [18F]EKZ-001 ([18F]Bavarostat), a radiotracer selective for HDAC6, in healthy adult subjects. METHODS: Biodistribution and radiation dosimetry studies were performed in four healthy subjects (2M/2F, 23.5 ± 2.4 years) using sequential whole-body PET/CT. The most appropriate kinetic model to quantify brain uptake was determined in 12 healthy subjects (6M/6F, 57.6 ± 3.7 years) from 120-min dynamic PET/MR scans using a radiometabolite-corrected arterial plasma input function. Four subjects underwent retest scans (2M/2F, 57.3 ± 5.6 years) with a 1-day interscan interval to determine test-retest variability (TRV). Regional volume of distribution (VT) was calculated using one-tissue and two-tissue compartment models (1-2TCM) and Logan graphical analysis (LGA), with time-stability assessed. VT differences between males and females were evaluated using volume of interest and whole-brain voxel-wise approaches. RESULTS: The effective dose was 39.1 ± 7.0 µSv/MBq. Based on the Akaike information criterion, 2TCM was the preferred model compared to 1TCM. Regional LGA VT were in agreement with 2TCM VT, however demonstrated a lower absolute TRV of 7.7 ± 4.9%. Regional VT values were relatively homogeneous with highest values in the hippocampus and entorhinal cortex. Reduction of acquisition time was achieved with a 0 to 60-min scan followed by a 90 to 120-min scan. Males demonstrated significantly higher VT than females in the majority of cortical and subcortical brain regions. No relevant radiotracer related adverse events were reported. CONCLUSION: [18F]EKZ-001 is safe and appropriate for quantifying HDAC6 expression in the human brain with Logan graphical analysis as the preferred quantitative approach. Males showed higher HDAC6 expression across the brain compared to females.

Cell proliferation detected using [18F]FLT PET/CT as an early marker of abdominal aortic aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a focal aortic dilatation progressing towards rupture. Non-invasive AAA-associated cell proliferation biomarkers are not yet established. We investigated the feasibility of the cell proliferation radiotracer, fluorine-18-fluorothymidine ([18F]FLT) with positron emission tomography/computed tomography (PET/CT) in a progressive pre-clinical AAA model (angiotensin II, AngII infusion). METHODS AND RESULTS: Fourteen-week-old apolipoprotein E-knockout (ApoE-/-) mice received saline or AngII via osmotic mini-pumps for 14 (n = 7 and 5, respectively) or 28 (n = 3 and 4, respectively) days and underwent 90-minute dynamic [18F]FLT PET/CT. Organs were harvested from independent cohorts for gamma counting, ultrasound scanning, and western blotting. [18F]FLT uptake was significantly greater in 14- (n = 5) and 28-day (n = 3) AAA than in saline control aortae (n = 5) (P < 0.001), which reduced between days 14 and 28. Whole-organ gamma counting confirmed greater [18F]FLT uptake in 14-day AAA (n = 9) compared to saline-infused aortae (n = 4) (P < 0.05), correlating positively with aortic volume (r = 0.71, P < 0.01). Fourteen-day AAA tissue showed increased expression of thymidine kinase-1, equilibrative nucleoside transporter (ENT)-1, ENT-2, concentrative nucleoside transporter (CNT)-1, and CNT-3 than 28-day AAA and saline control tissues (n = 3 each) (all P < 0.001). CONCLUSIONS: [18F]FLT uptake is increased during the active growth phase of the AAA model compared to saline control mice and late-stage AAA.

Silencing microRNA-330-5p increases MMP1 expression and promotes an invasive phenotype in oesophageal adenocarcinoma.

BACKGROUND: Many patients diagnosed with oesophageal adenocarcinoma (OAC) present with advanced disease and approximately half present with metastatic disease. Patients with localised disease, who are managed with curative intent, frequently undergo neoadjuvant chemoradiotherapy. Unfortunately, ~ 70% of patients have little or no response to chemoradiotherapy. We previously identified miR-330-5p as being the most significantly downregulated microRNA in the pre-treatment OAC tumours of non-responders to treatment, but that loss of miR-330-5p had a limited impact on sensitivity to chemotherapy and radiation in vitro. Here, we further examined the impact of miR-330-5p loss on OAC biology. METHODS: miR-330-5p was suppressed in OE33 OAC cells following stable transfection of a vector-driven anti-sense RNA. Whole transcriptome digital RNA-Seq was employed to identify miR-330-5p regulated genes, and qPCR was used for validation. Protein expression was assessed by protein array, Western blotting and zymography. Invasive potential was measured using a transwell assay system. Tumour xenograft growth profile studies were performed in immunocompromised CD1 mice. RESULTS: In OE33 cells, suppression of miR-330-5p significantly altered expression of 42 genes, and several secreted proteases. MMP1 gene expression and protein secretion was significantly enhanced with miR-330-5p suppression. This corresponded to enhanced collagen invasion in vitro. In vivo, OE33-derived tumour xenografts with miR-330-5p suppression grew faster than controls. CONCLUSIONS: Loss of miR-330-5p expression in OAC tumours may influence tumour cell invasive capacity, tumour growth and therapeutic sensitivity via alterations to the tumour microenvironment.

Visualizing Kinetically Robust CoIII4L6 Assemblies in Vivo: SPECT Imaging of the Encapsulated [99mTc]TcO4- Anion.

Noncovalent encapsulation is an attractive approach for modifying the efficacy and physiochemical properties of both therapeutic and diagnostic species. Abiotic self-assembled constructs have shown promise, yet many hurdles between in vitro and (pre)clinical studies remain, not least the challenges associated with maintaining the macromolecular, hollow structure under nonequilibrium conditions. Using a kinetically robust CoIII4L6 tetrahedron we now show the feasibility of encapsulating the most widely used precursor in clinical nuclear diagnostic imaging, the γ-emitting [99mTc]TcO4- anion, under conditions compatible with in vivo administration. Subsequent single-photon emission computed tomography imaging of the caged-anion reveals a marked change in the biodistribution compared to the thyroid-accumulating free oxo-anion, thus moving clinical applications of (metallo)supramolecular species a step closer.

WSB-1 regulates the metastatic potential of hormone receptor negative breast cancer.

BACKGROUND: Metastatic spread is responsible for the majority of cancer-associated deaths. The tumour microenvironment, including hypoxia, is a major driver of metastasis. The aim of this study was to investigate the role of the E3 ligase WSB-1 in breast cancer biology in the context of the hypoxic tumour microenvironment, particularly regarding metastatic spread. METHODS: In this study, WSB-1 expression was evaluated in breast cancer cell lines and patient samples. In silico analyses were used to determine the impact of WSB-1 expression on distant metastasis-free survival (DMFS) in patients, and correlation between WSB1 expression and hypoxia gene expression signatures. The role of WSB-1 on metastasis promotion was evaluated in vitro and in vivo. RESULTS: High WSB1 expression was associated with decreased DMFS in ER-breast cancer and PR-breast cancer patients. Surprisingly, WSB1 expression was not positively correlated with known hypoxic gene expression signatures in patient samples. Our study is the first to show that WSB-1 knockdown led to decreased metastatic potential in breast cancer hormone receptor-negative models in vitro and in vivo. WSB-1 knockdown was associated with decreased metalloproteinase (MMP) activity, vascular endothelial growth factor (VEGF) secretion, and angiogenic potential. CONCLUSIONS: Our data suggests that WSB-1 may be an important regulator of aggressive metastatic disease in hormone receptor-negative breast cancer. WSB-1 could therefore represent a novel regulator and therapeutic target for secondary breast cancer in these patients.

A practical microwave method for the synthesis of fluoromethy 4-methylbenzenesulfonate in tert-amyl alcohol.

Fluorine substitution is an established tool in medicinal chemistry to favourably alter the molecular properties of a lead compound of interest. However, gaps still exist in the library of synthetic methods for accessing certain fluorine-substituted motifs. One such area is the fluoromethyl group, particularly when required in a fluoroalkylating capacity. The cold fluorination of methylene ditosylate is under evaluated in the literature, often proceeding with low yields or harsh conditions. This report describes a novel microwave method for the rapid nucleophilic fluorination of methylene ditosylate using inexpensive reagents in good isolated yield (65%).

Multimodal nanoparticle imaging agents: design and applications.

Molecular imaging, where the location of molecules or nanoscale constructs can be tracked in the body to report on disease or biochemical processes, is rapidly expanding to include combined modality or multimodal imaging. No single imaging technique can offer the optimum combination of properties (e.g. resolution, sensitivity, cost, availability). The rapid technological advances in hardware to scan patients, and software to process and fuse images, are pushing the boundaries of novel medical imaging approaches, and hand-in-hand with this is the requirement for advanced and specific multimodal imaging agents. These agents can be detected using a selection from radioisotope, magnetic resonance and optical imaging, among others. Nanoparticles offer great scope in this area as they lend themselves, via facile modification procedures, to act as multifunctional constructs. They have relevance as therapeutics and drug delivery agents that can be tracked by molecular imaging techniques with the particular development of applications in optically guided surgery and as radiosensitizers. There has been a huge amount of research work to produce nanoconstructs for imaging, and the parameters for successful clinical translation and validation of therapeutic applications are now becoming much better understood. It is an exciting time of progress for these agents as their potential is closer to being realized with translation into the clinic. The coming 5-10 years will be critical, as we will see if the predicted improvement in clinical outcomes becomes a reality. Some of the latest advances in combination modality agents are selected and the progression pathway to clinical trials analysed.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

Detection of apoptosis by PET/CT with the diethyl ester of [¹8F]ML-10 and fluorescence imaging with a dansyl analogue.

The diethyl ester of [(18)F]ML-10 is a small molecule apoptotic PET probe for cancer studies. Here we report a novel multi-step synthesis of the diethyl ester of ML-10 in excellent yields via fluorination using Xtal-Fluor-E. In addition, a one-pot radiosynthesis of the diethyl ester of [(18)F]ML-10 from nucleophilic [(18)F]fluoride was completed in 23% radiochemical yield (decay corrected). The radiochemical purity of the product was ≥99%. The diethyl ester of [(18)F]ML-10 was used in vivo to detect apoptosis in the testes of mice. In parallel studies, the dansyl-ML-10 diethyl ester was prepared and used to detect apoptotic cells in an in vitro cell based assay.

Direct comparison of [18F]AlF-NOTA-JR11 and [18F]AlF-NOTA-octreotide for PET imaging of neuroendocrine tumors: Antagonist versus agonist.

BACKGROUND: [18F]AlF-NOTA-octreotide is an 18F-labeled somatostatin analogue which is a good clinical alternative for 68Ga-labeled somatostatin analogues. However, radiolabeled somatostatin receptor (SSTR) antagonists might outperform agonists regarding imaging sensitivity of neuroendocrine tumors (NETs). No direct comparison between the antagonist [18F]AlF-NOTA-JR11 and the agonist [18F]AlF-NOTA-octreotide as SSTR PET probes is available. Herein, we present the radiosynthesis of [18F]AlF-NOTA-JR11 and compare its NETs imaging properties directly with the established agonist radioligand [18F]AlF-NOTA-octreotide preclinically. METHODS: [18F]AlF-NOTA-JR11 was synthesized in an automated synthesis module. The in vitro binding characteristics (IC50) of [natF]AlF-NOTA-JR11 and [natF]AlF-NOTA-octreotide were evaluated and the in vitro stability of [18F]AlF-NOTA-JR11 was determined in human serum. In vitro cell binding and internalization was performed with [18F]AlF-NOTA-JR11 and [18F]AlF-NOTA-octreotide using SSTR2 expressing cells and the pharmacokinetics were evaluated using µPET/CT in mice bearing BON1.SSTR2 tumor xenografts. RESULTS: Excellent binding affinity for SSTR2 was found for [natF]AlF-NOTA-octreotide (IC50 of 25.7 ± 7.9 nM). However, the IC50 value for [natF]AlF-NOTA-JR11 (290.6 ± 71 nM) was 11-fold higher compared to [natF]AlF-NOTA-octreotide, indicating lower affinity for SSTR2. [18F]AlF-NOTA-JR11 was obtained in a good RCY (50 ± 6 %) but with moderate RCP of 94 ± 1 %. [18F]AlF-NOTA-JR11 demonstrated excellent stability in human serum (>95 % after 240 min). 2.7-fold higher cell binding was observed for [18F]AlF-NOTA-JR11 as compared to [18F]AlF-NOTA-octreotide after 60 min. µPET/CT images demonstrated comparable pharmacokinetics and tumor uptake between [18F]AlF-NOTA-JR11 (SUVmax: 3.7 ± 0.8) and [18F]AlF-NOTA-octreotide (SUVmax: 3.6 ± 0.4). CONCLUSIONS: [18F]AlF-NOTA-JR11 was obtained in good RCY, albeit with a moderate RCP. The cell binding study showed significant higher binding of [18F]AlF-NOTA-JR11 compared to [18F]AlF-NOTA-octreotide, despite the higher IC50 value of AlF-NOTA-JR11. However, pharmacokinetics and in vivo tumor uptake was comparable for both radiotracers. Novel Al18F-labeled derivatives of JR11 with higher SSTR2 affinity should be developed for increased tumor uptake and NET imaging sensitivity.