Ubiquitylation and ISGylation are protein post-translational modifications (PTMs) and two of the main events involved in the activation of pattern recognition receptor (PRRs) signals allowing the host defense response to viruses. As with similar viruses, SARS-CoV-2, the virus causing COVID-19, hijacks these pathways by removing ubiquitin and/or ISG15 from proteins using a protease called PLpro, but also by interacting with enzymes involved in ubiquitin/ISG15 machinery. These enable viral replication and avoidance of the host immune system. In this review, we highlight potential points of therapeutic intervention in ubiquitin/ISG15 pathways involved in key host\u2013pathogen interactions, such as PLpro, USP18, TRIM25, CYLD, A20, and others that could be targeted for the treatment of COVID-19, and which may prove effective in combatting current and future vaccine-resistant variants of the disease.
\n \n\n \n \nBackground: Abdominal aortic aneurysms (AAA) are pathological dilatations of the aorta which can result in rupture and mortality. Novel methods of predicting AAA growth is a recognised priority in AAA research. Patient with AAAs have increased risk of cardiovascular morbidity. We have previously observed accelerated systemic endothelial dysfunction (measured by brachial artery FMD) in AAA patients and FMD correlates with future AAA growth. Further, systemic endothelial dysfunction is reversed by AAA repair. AAAs contain intra-luminal thrombus (ILT). Since ILT is either removed or excluded from circulation after successful repair of AAAs, we hypothesise that ILT to be the source of mediators that contribute to AAA growth. Methods: Patients were prospectively recruited to the Study (Ethics Ref SC/13/0250). Plasma samples were collected at baseline and at 1 year from each patient. Plasma samples were also collected before and at 10-12 weeks after surgery from each patient (n=29). Paired aneurysm wall, ILT, omental biopsies were collected intra-operatively during open surgical repair (n=3). In addition to analyses of the tissue, supernatant was obtained from ex vivo culture of these paired tissue samples. Samples were subjected to non-targeted LC-MSMS workflow after trypsin digest, using the Universal method to discover novel proteins. LC-MSMS data was analysed using the Progenesis QI pipeline. Results: The median AAA size at baseline was 48 mm. 59 patients were prospectively followed for 12 months. The median growth rate of AAA was 3.8%/year (IQR 1.9% to 6.8%). Comparison between patients with the fastest vs the slowest (n=10 each) showed 116 proteins to be differentially expressed in their plasma. Among these proteins, 35 also changed significantly before and after AAA repair, suggesting their origin to from the AAA complex. Comparison of the proteomics profile of aneurysm tissue, ILT, and omental artery show 128 proteins to be uniquely present in ILT. Analyses of the tissue culture supernatant further revealed 3 proteins that are: (i) uniquely present in ILT; (ii) released by ILT; (iii) systemic levels reduced after AAA surgery; (iv) differs between fast and slow growth AAAs. One of these proteins is attractin. To validate the LC-MSMS data, attractin level in individual patient was measured by ELISA. Consistent with the LC-MSMS data, plasma attractin level is higher in patients with fast AAA growth. Plasma attractin level correlates significantly with future AAA growth rate (Spearman r=0.35, P<;0.005). Using attractin and AAA diameter as input variables, the AUROC for predicting no growth of AAA at 12 months is 85% (P<0.001). Conclusion: We show that ILT of AAAs releases mediators (such as attractin) during the natural history of AAA growth. These are novel biomarkers for AAA growth prediction in humans.
\n \n\n \n \nAbstractT cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. Additionally, Th2 polarization digresses in the absence of CCT-controlled protein folding resulting paradoxically in continued IFN-\u03b3 expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.
\n \n\n \n \nIschemia-reperfusion injury (IRI) is a hallmark for tissue injury in donation after circulatory death (DCD) kidneys. The implementation of hypothermic machine perfusion (HMP) provides a platform for improved preservation of DCD kidneys. Doxycycline administration has shown protective effects during IRI. Therefore, we explored the impact of doxycycline on proteolytic degradation mechanisms and the urinary proteome of perfused kidney grafts. Porcine kidneys underwent 30 min of warm ischemia, 24 h of oxygenated HMP (control/doxycycline) and 240 min of ex vivo reperfusion. A proteomic analysis revealed distinctive clustering profiles between urine samples collected at T15 min and T240 min. High-efficiency undecanal-based N-termini (HUNTER) kidney tissue degradomics revealed significantly more proteolytic activity in the control group at T-10. At T240, significantly more proteolytic activity was observed in the doxycycline group, indicating that doxycycline alters protein degradation during HMP. In conclusion, doxycycline administration during HMP led to significant proteomic and proteolytic differences and protective effects by attenuating urinary NGAL levels. Ultimately, we unraveled metabolic, and complement and coagulation pathways that undergo alterations during machine perfusion and that could be targeted to attenuate IRI induced injury.
\n \n\n \n \nEnhanced drug release and bioavailability of poorly soluble active pharmaceutical ingredient (API) can be achieved via a fluidized bed coating integrated with supercritical anti-solvent (SAS-FB) - a process of precipitating drug particles onto carrier granules. However, in the absence of excipients, SAS-FB often results in crystalline of the API on the surface of carriers, limiting the improvement of pharmaceutical properties. Co-processing with excipients is considered an effective approach to improve drug release in the SAS-FB process. Our study used sirolimus, an immune suppressive agent, as the model API to characterize excipients for their effect on pharmaceutical properties in the SAS-FB process. We show that co-precipitation of excipients and sirolumus impacts on carrier specific surface area and drug yield. Among the tested excipients, formulation containing polyvinylpyrrolidone K30 achieved the highest drug yield. Importantly, compared with Rapamune\u00ae tablet, our optimized formulation displayed a superior in vivo oral bioavailability by 3.05-fold in Sprague-Dawley rats and 3.99-fold in beagle dogs. A series of characterization of the processed API was performed to understand the mechanism by which excipients contributed to drug dissolution properties. Our study provides a useful guidance for the use of excipients in the SAS-FB technology to improve pharmaceutical properties of sirolimus and other poorly soluble drugs.
\n \n\n \n \nThe COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global crisis with unprecedented challenges for public health. Vaccinations against SARS-CoV-2 have slowed the incidence of new infections and reduced disease severity. As the time of day of vaccination has been reported to influence host immune responses to multiple pathogens, we quantified the influence of SARS-CoV-2 vaccination time, vaccine type, participant age, sex, and days post-vaccination on anti-Spike antibody responses in health care workers. The magnitude of the anti-Spike antibody response is associated with the time of day of vaccination, vaccine type, participant age, sex, and days post-vaccination. These results may be relevant for optimising SARS-CoV-2 vaccine efficacy.
\n \n\n \n \nAbstractNP105\u2013113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105\u2013113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n\u2009=\u200977, convalescent n\u2009=\u200952). We demonstrated a beneficial association of NP105\u2013113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105\u2013113-B*07:02-specific T cells were maintained 6\u2009months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105\u2013113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.
\n \n\n \n \nDespite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.
\n \n\n \n \nHepatitis C virus (HCV) is highly variable and transmits through infected blood to establish a chronic liver infection in the majority of patients. Our knowledge on the infectivity of clinical HCV strains is hampered by the lack of in vitro cell culture systems that support efficient viral replication. We and others have reported that HCV can associate with and infect immune cells and may thereby evade host immune surveillance and elimination. To evaluate whether B cells play a role in HCV transmission, we assessed the ability of B cells and sera from recent (<2 years) or chronic (\u2265 2 years) HCV patients to infect humanized liver chimeric mice. HCV was transmitted by B cells from chronic infected patients whereas the sera were non-infectious. In contrast, B cells from recently infected patients failed to transmit HCV to the mice, whereas all serum samples were infectious. We observed an association between circulating anti-glycoprotein E1E2 antibodies and B cell HCV transmission. Taken together, our studies provide evidence for HCV transmission by B cells, findings that have clinical implications for prophylactic and therapeutic antibody-based vaccine design.
\n \n\n \n \nAbstractPersistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment.
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