Site-specific steric control of SARS-CoV-2 spike glycosylation

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

The SARS-CoV-2 pandemic is continuing to disrupt personal lives, global healthcare systems and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits and cynomolgus macaques. The vaccine-induced immunity protected macaques against a high dose challenge, resulting in strongly reduced viral infection and replication in upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Serological responses to SARS-CoV-2 following non-hospitalised infection: clinical and ethnodemographic features associated with the magnitude of the antibody response

ObjectiveTo determine clinical and ethnodemographic correlates of serological responses against the SARS-CoV-2 spike glycoprotein following mild-to-moderate COVID-19. DesignA retrospective cohort study of healthcare workers who had self-isolated due to COVID-19. SettingUniversity Hospitals Birmingham NHS Foundation Trust, UK (UHBFT). Participants956 health care workers were recruited by open invitation via UHBFT trust email and social media. InterventionParticipants volunteered a venous blood sample that was tested for the presence of anti-SARS-CoV-2 spike glycoprotein antibodies. Results were interpreted in the context of the symptoms of their original illness and ethnodemographic variables. ResultsUsing an assay that simultaneously measures the combined IgG, IgA and IgM response against the spike glycoprotein (IgGAM), the overall seroprevalence within this cohort was 46.2% (n=442/956). The seroprevalence of immunoglobulin isotypes was 36.3%, 18.7% and 8.1% for IgG, IgA and IgM respectively. IgGAM identified serological responses in 40.6% (n=52/128) of symptomatic individuals who reported a negative SARS-CoV-2 PCR test. Increasing age, non-white ethnicity and obesity were independently associated with greater IgG antibody response against the spike glycoprotein. Self-reported fever and fatigue were associated with greater IgG and IgA responses against the spike glycoprotein. The combination of fever and/or cough and/or anosmia had a positive predictive value of 92.3% for seropositivity. Conclusions and relevanceAssays employing combined antibody detection demonstrate enhanced seroepidemiological sensitivity and can detect prior viral exposure even when PCR swabs have been negative. We demonstrate an association between known ethnodemographic risk factors associated with mortality from COVID-19 and the magnitude of serological responses in mild-to-moderate disease. The combination of cough, and/or fever and/or anosmia identifies the majority of individuals who should self-isolate for COVID-19.

Sensitive detection of SARS-CoV-2-specific-antibodies in dried blood spot samples

ImportancePopulation-wide serological testing is an essential component in understanding the COVID-19 pandemic. The logistical challenges of undertaking widespread serological testing could be eased through use of a reliable dried blood spot (DBS) sampling method. ObjectiveTo validate the use of dried blood spot sampling for the detection of SARS-CoV-2-specific antibodies. Design, setting and participantsEighty-seven matched DBS and serum samples were obtained from eighty individuals, including thirty-one who were previously PCR-positive for SARS-CoV-2. DBS eluates and sera were used in an ELISA to detect antibodies to the viral spike protein. ResultsSpecific anti-SARS-Cov-2 spike glycoprotein antibodies were detectable in both serum and DBS eluate and there was a significant correlation between the antibody levels detected in matched samples (r = 0.96, p<0.0001). Using serum as the gold standard in the assay, matched DBS samples achieved a Cohens kappa coefficient of 0.975 (near-perfect agreement), a sensitivity of 98.1% and specificity of 100%, for detecting anti-spike glycoprotein antibodies. Conclusions and relevanceEluates from DBS samples are a reliable and reproducible source of antibodies to be used for the detection of SARS-CoV-2-specific antibodies. The use of DBS sampling could complement the use of venepuncture in the immunosurveillance of COVID-19 in both low and high income settings.

Serology confirms SARS-CoV-2 infection in PCR-negative children presenting with Paediatric Inflammatory Multi-System Syndrome

BackgroundDuring the COVID-19 outbreak, reports have surfaced of children who present with features of a multisystem inflammatory syndrome with overlapping features of Kawasaki disease and toxic shock syndrome - Paediatric Inflammatory Multisystem Syndrome-temporally associated with SARS-CoV-2 pandemic (PIMS-TS). Initial reports find that many of the children are PCR-negative for SARS-CoV-2, so it is difficult to confirm whether this syndrome is a late complication of viral infection in an age group largely spared the worst consequences of this infection, or if this syndrome reflects enhanced surveillance. MethodsChildren hospitalised for symptoms consistent with PIMS-TS between 28 April and 8 May 2020, and who were PCR-negative for SARS-CoV-2, were tested for antibodies to viral spike glycoprotein using an ELISA test. ResultsEight patients (age range 7-14 years, 63% male) fulfilled case-definition for PIMS-TS during the study period. Six of the eight patients required admission to intensive care. All patients exhibited significant IgG and IgA responses to viral spike glycoprotein. Further assessment showed that the IgG isotypes detected in children with PIMS-TS were of the IgGl and lgG3 subclasses, a distribution similar to that observed in samples from hospitalised adult COVID-19 patients. In contrast, lgG2 and lgG4 were not detected in children or adults. IgM was not detected in children, which contrasts with adult hospitalised adult COVID-19 patients of whom all had positive IgM responses. ConclusionsStrong IgG antibody responses can be detected in PCR-negative children with PIMS-TS. The low detection rate of IgM in these patients is consistent with infection having occurred weeks previously and that the syndrome onset occurs well after the control of SARS-CoV-2 viral load. This implies that the disease is largely immune-mediated. Lastly, this indicates that serology can be an appropriate diagnostic tool in select patient groups.

Detection of antibodies to the SARS-CoV-2 spike glycoprotein in both serum and saliva enhances detection of infection

BackgroundDetecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. MethodsWe systemically developed an ELISA assay, optimising different antigens and amplification steps, in serum and saliva from symptomatic and asymptomatic SARS-CoV-2-infected subjects. ResultsUsing trimeric spike glycoprotein, rather than nucleocapsid enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike, but not nucleocapsid, IgG, IgA and IgM antibody responses were readily detectable in saliva from non-hospitalized symptomatic and asymptomatic individuals. Antibody responses in saliva and serum were largely independent of each other and symptom reporting. ConclusionsDetecting antibody responses in both saliva and serum is optimal for determining virus exposure and understanding immune responses after SARS-CoV-2 infection. FundingThis work was funded by the University of Birmingham, the National Institute for Health Research (UK), the NIH National Institute for Allergy and Infectious Diseases, the Bill and Melinda Gates Foundation and the University of Southampton.

SARS-CoV-2 seroconversion in health care workers

BackgroundThe correlates of protection against SARS-CoV-2 and their longevity remain unclear. Studies in severely ill individuals have identified robust cellular and humoral immune responses against the virus. Asymptomatic infection with SARS-CoV-2 has also been described, but it is unknown whether this is sufficient to produce antibody responses. MethodsWe performed a cross-sectional study recruiting 554 health care workers from University Hospitals Birmingham NHS Foundation Trust who were at work and asymptomatic. Participants were tested for current infection with SARS-CoV-2 by nasopharyngeal swab for real-time polymerase chain reaction and for seroconversion by the measurement of anti-SARS-CoV-2 spike glycoprotein antibodies by enzyme linked immunosorbent assay. Results were interpreted in the context of previous, self-reported symptoms of illness consistent with COVID-19. ResultsThe point prevalence of infection with SARS-CoV-2, determined by the detection of SARS-CoV-2 RNA on nasopharnygeal swab was 2.39% (n=13/544). Serum was available on 516 participants. The overall rate of seroconversion in the cohort was 24.4% (n=126/516). Individuals who had previously experienced a symptomatic illness consistent with COVID-19 had significantly greater seroconversion rates than those who had remained asymptomatic (37.5% vs 17.1%, {chi}2 =21.1034, p<0.0001). In the week preceding peak COVID-19-related mortality at UHBFT, seroconversion rates amongst those who were suffering from symptomatic illnesses peaked at 77.8%. Prior symptomatic illness generated quantitatively higher antibody responses than asymptomatic seroconversion. Seroconversion rates were highest amongst those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%) with lower rates observed in participants working in intensive care (14.8%) and emergency medicine (13.3%). ConclusionsIn a large cross-sectional seroprevalence study of health-care workers, we demonstrate that asymptomatic seroconversion occurs, however prior symptomatic illness is associated with quantitatively higher antibody responses. The identification that the potential for seroconversion in health-care workers can associate differentially with certain hospital departments may inform future infection control and occupational health practices. Research in contextO_ST_ABSEvidence before the studyC_ST_ABSTo date, no study has examined the cross-sectional seroprevalence of anti-SARS-CoV-2 antibodies in health care workers during the COVID-19 pandemic. Existing evidence suggests that the levels of SARS-CoV-2 antibodies developing following infection may vary with disease severity in keeping with previous coronavirus pandemics. Added value of this studyWe demonstrate that seroconversion can occur in health care workers who have suffered no previous symptoms of SARS-Cov-2 infection. However, prior symptomatic infection tends to drive quantitatively superior antibody responses against the virus. We observed differential seroconversion rates in individuals working within different hospital departments. Using intensive care as a reference, the relative risk for seroconversion was greatest for those working in housekeeping, acute and general internal medicine. Implications of all the available evidenceInsight into the current seroprevalence of SARS-CoV-2 antibodies within a high-risk cohort of health-care workers is of direct relevance as a reference point for future community serological surveys. We provide further evidence of asymptomatic infection and seroconversion, strengthening the argument for regular, routine screening of health-care workers. Finally, we provide evidence that individuals working in particular roles within the NHS are at greater risk of seroconversion with significant implications for their occupational health.

Site-specific analysis of the SARS-CoV-2 glycan shield

The emergence of the betacoronavirus, SARS-CoV-2 that causes COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, that mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in immune evasion and occluding immunogenic protein epitopes. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

Vulnerabilities in coronavirus glycan shields despite extensive glycosylation

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development has focussed on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein, which mediates receptor recognition and membrane fusion. Coronavirus S proteins are extensively glycosylated viral fusion proteins, encoding around 69-87 N-linked glycosylation sites per trimeric spike. Using a multifaceted structural approach, we reveal a specific area of high glycan density on MERS S that results in the formation of under-processed oligomannose-type glycan clusters, which was absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Consistent with the ability of the antibody-mediated immune response to effectively target and neutralize coronaviruses, we demonstrate that the glycans of coronavirus spikes are not able to form an efficacious high-density global shield to thwart the humoral immune response. Overall, our data reveal how differential organisation of viral glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the viral protein surface.

Treatment and Outcome of Acute Aortic Dissection Associated with Atherosclerotic Aortic Aneurysm / 日本心臓血管外科学会雑誌

We set out to assess our treatment strategy of acute aortic dissection associated with atherosclerotic aortic aneurysm, and to assess its results. A total of 228 patients with acute aortic dissection were admitted to our hospital between 1994 and 2009. Among these, 30 cases were associated with atherosclerotic aortic aneurysm and we retrospectively analyzed their patient data. Of these, 5 patients received diagnoses of Stanford A dissection and 25 patients demonstrated Stanford B. Coexisting aneurysms consisted of postabdominal aortic replacement in 9 patients, ascending aortic aneurysm in 1, arch aortic aneurysm in 6, descending aortic aneurysm in 2, thoracoabdominal aortic aneurysm in 3, and abdominal aortic aneurysm in 9. Patients were divided into 3 groups based on the relationship between aneurysm and dissection : acute aortic dissection occurred after graft replacement of an aortic aneurysm (Group 1, <i>n</i>=9), dissection coexisted with aneurysm in a different segment of the aorta (Group 2, <i>n</i>=8), and dissection extended to or involved the aneurysm (Group 3, <i>n</i>=13). Our treatment strategy for all patients excluding those with aortic rupture or malperfusion is described below. In the cases of Stanford A dissection, emergency ascending aortic replacement or total arch replacement was performed. In cases of Stanford B, patients were treated conservatively in the acute phase. Surgery for the coexisting aortic aneurysm was then performed in the chronic phase, if the aneurysm was large. We think that the interval between dissection onset and aneurysm surgery should be extended to at least 1 month, because the aortic wall was too fragile to perform anastomosis in the acute phase in the present cases. As a result, there were 2 hospital deaths in Group 3, but there was no aortic rupture during treatment in the acute phase in any of these 3 groups. There were no vascular-related deaths during follow up. Our treatment strategy obtained favorable outcomes.

A case of Aortic Valve Endocarditis with Splenic Abscess / 日本心臓血管外科学会雑誌

A 25-year-old man was admitted with high fever and heart murmur. Echocardiogram showed left ventricular chamber dilatation and vegetations attached to the aortic valve. Blood cultures obtained on admission revealed <i>Streptococcus viridans</i>. Despite adequate antibiotic therapy, congestive heart failure progressively worsened and large splenic abscesses were detected by computed tomography. Urgent aortic valve replacement and splenectomy were performed. The aortic valve was bicuspid and markedly destroyed. Pathology of the spleen showed findings consistent with large infarct and abscesses due to septic emboli. The postoperative course was uneventful.

Autologous Blood Donation in Open-Heart Surgery in Cooperation with the Red Cross Blood Center / 日本心臓血管外科学会雑誌

An investigation on the efficacy of preoperative autologous blood donation in open-heart surgery was made using frozen red blood cells and MAP red blood cells in cooperation with the Red Cross Blood Center. In 109 cases which received the donation, the rate of cases which received no homologous blood transfusion was 93.6% (35.3% in the cases without donation). Even in the cases of redo operation or aortic surgery, in which extensive blood loss is expected, 75% of those given a donation of 1600-2000ml frozen blood required no homologous blood transfusion. The hemoglobin concentration in the cases which received blood donation for more than 4 weeks did not decrease, indicating that safe donation is feasible. The aforementioned frozen and MAP blood preparations can be preserved for a long period so that blood donation can be started even before deciding on the date of operation. Also, its usefulness is not affected by the postponement of the operation. Furthermore, there was no problem in safety with respect to transfer, treatment, and storage of the autologous blood in cooperation with the Red Cross Blood Center, suggesting that this is useful as a preoperative donation method, especially in small- and middle-scale hospitals, which have no separate blood centers. However, there were 2 cases in which aggravated symptoms were noted after blood collection. Therefore, it is important to carefully select cases for autologous blood donation in open-heart surgery and it is desirable to set up appropriate donation schedules.

A Case Report of Abdominal Aortic Aneurysm with Isolated Left-side Inferior Vena Cava / 日本心臓血管外科学会雑誌

Isolated left-side inferior vena cava is rare, there being only four cases associated with abdominal aortic aneurysm reported so far in the Japanese literature. A 72-year-old man was admitted to our hospital for the evaluation of an abdominal pulsatile mass. CT scan revealed abdominal aortic aneurysm with isolated left-sided inferior vena cava. Aneurysmectomy and bifurcated graft replacement was performed with retracting inferior vena cava. The postoperative course was uneventful.

Effectiveness of Erythropoietin in Elderly Coronary Bypass Patients / 日本心臓血管外科学会雑誌

The effectiveness of recombinant human erythropoietin (rHuEPO) was evaluated in elderly patients who underwent coronary artery bypass grafting. A total of 133 patients were divided into three groups: those who were 70 years of age or older and received rHuEPO (group I; <i>n</i>=32), those who were also 70 years of age or older but did not receive rHuEPO (group II; <i>n</i>=35), and those who were 60 years or younger and received rHuEPO (group III; <i>n</i>=66). In 87.5% of group I, 42.9% of group II, and 98.5% of group III, homologous blood transfusion could be avoided. The percentage of patients without homologous blood transfusion was significantly higher in group I than in group II (<i>p</i><0.001). The rate of homologous blood transfusion was significantly higher in group I than in group III (<i>p</i><0.05), but rHuEPO had equal effects in terms of increase in hemoglobin level in the two groups. Furthermore, in patients without anemia, the rate of homologous blood transfusion was almost the same in the two groups. In conclusion, the administration of rHuEPO enables even elderly patients to undergo coronary artery bypass grafting without homologous blood transfusion.

Coronary Artery Bypass Grafting in Patients with Severe Calcified Ascending Aorta with Aortic No-touch Technique / 日本心臓血管外科学会雑誌

Coronary artery bypass grafting using hypothermic circulatory arrest and ventricular fibrillation without aortic cross clamping in 6 patients with severely calcified aortas is described. The use of hypothermic circulatory arrest or ventricular fibrillation has not been established in coronary artery bypass grafting. We recently used aortic no-touch technique in 6 patients. All patients were supported and cooled with cardiopulmonary bypass, and circulatory arrest was performed in 3 patients. With the exception of one hemodialysis patient, 5 patients survived without neurological deficit. We think the aortic no-touch technique is safe and reliable in coronary artery bypass grafting with severe calcified aortas.

Perioperative Cerebral Infarction during or after Coronary Artery Bypass Grafting / 日本心臓血管外科学会雑誌

A total of 961 patients underwent coronary artery bypass grafting (CABG) between 1982 and 1991, and we investigated perioperative cerebral infarction. The average age of operation in these case was 65±4 years. There was 9 patients with hypertension, 7 with diabetes mellitus and 5 with hyperlipidemia. Concerning cerebral infarction, there were 3 patients with multiple infarction, 6 with infarction of the mid cerebral artery area, 1 with infarction of posterior cerebral artery area, 1 with infarction of posterior cerebral artery area, 1 with infarction of pons and 1 with infarction of the ophthalmic artery. The courses of infarction involved atherosclerosis, hypoperfusion during cardiopulmonary bypass, thrombosis due to arterial fibrillation and thrombus on the left ventricular wall. Three patients who had critical cerebral infarction died after CABG. We consider that avoid perioperative cerebral infarction preoperative atherosclerosis, thrombus and to choose the proper procedure of the operation.

A Case Report of Coronary Artery Bypass Grafting with Idiopathic Interstitial Pneumonia / 日本心臓血管外科学会雑誌

A 72 year-old man underwent coronary angiography (CAG) with a diagnosis of unstable angina pectoris, and 90% stenosis of the LMT was found. Since idiopathic interstitial pneumonia (IIP) had been diagnosed previously, percutaneous transluminal coronary angioplasty (PTCA) was performed. However, his unstable angina recurred after about 2 months restenosis of the LMT to 90% was shown by CAG, and coronary artery bypass grafting (CABG) was performed. In the preoperative chest X-ray, diffuse granular opacities were seen in both lower lungfields, and Velcro rales were heard by ausculation. A spirogram could not be obtained because of his unstable angina, but the PaO₂ was a reasonable 70mmHg when breathing room air. In consideration of the age of the patient, a double coronary artery bypass grafting using a saphenous vein graft (SVG) was performed to minimize duration of anesthesia. His PaO₂ showed a transient decrease after the end of cardiopulmonary bypass (CPB), but the perioperative hemodynamics and respiratory status were stable and extubation was performed on the 1st postoperative day. No aggravation of his IIP occurred postoperatively and he was discharged on the 29th postoperative day.

Coronary Revascularization in a Patient with Calcified Aorta Using Ventricular Fibrillation without Aortic Cross-clamping / 日本心臓血管外科学会雑誌

Coronary artery bypass surgery in a 54-year-old female with severe calcified ascending aorta was performed with aortic no touch technique, Extracorporeal circulation with femoral cannulation was performed, and bilateral internal thoracic acteries and gastroepiploic artery were used as grafts under ventricular fibrillation and hypothermia without aortic cross-clamping. No neurological complication was observed and postoperative course was uneventful. We think the aortic no touch technique is safe and reliable in the coronary revascularization with severe calcified aorta.

Bilateral Atrioventricular Valve Replacement for a Case of Corrected Transposition of the Great Arteris - A Case Report / 日本心臓血管外科学会雑誌

A case of 38-year-old woman with corrected transposition of great arteries is reported. She was admitted for acute cardiac failure caused by not only the left-side atrioventricular regurgitation for the ruptured chordae tendineae, but also the right-side one. We have to perform double valve replacement emergently due to the progression of biventricular failure. Very few reports have described a surgical repair of the right-sided valve replacement. The postoperative course was favorable.