The SARS-CoV-2 Antibody and Drug Research Based on the Structure of the Spike Protein and the Mechanism of Cell Entry.

COVID-19 is a severe acute respiratory syndrome caused by a novel coronavirus, SARS-CoV- 2.COVID-19 is now a pandemic, and is not yet fully under control.As the surface spike protein (S) mediates the recognition between the virus and cell membrane and the process of cell entry, it plays an important role in the course of disease transmission.The study on the S protein not only elucidates the structure and function of virus-related proteins and explains their cellular entry mechanism, but also provides valuable information for the prevention, diagnosis and treatment of COVII)-19.Concentrated on the S protein of SARS-CoV-2, this review covers four aspects: (1 ) The structure of the S protein and its binding with angiotensin converting enzyme II (ACE2) , the specific receptor of SARS-CoV-2, is introduced in detail.Compared with SARS-CoV, the receptor binding domain (RBD) of the SARS-CoV- 2 S protein has a higher affinity with ACE2, while the affinity of the entire S protein is on the contrary.(2) Currently, the cell entry mechanism of SARS-CoV-2 meditated by the S protein is proposed to include endosomal and non-endosomal pathways.With the recognition and binding between the S protein and ACE2 or after cell entry, transmembrane protease serine 2(TMPRSS2) , lysosomal cathepsin or the furin enzyme can cleave S protein at S1/S2 cleavage site, facilitating the fusion between the virus and target membrane.(3) For the progress in SARS-CoV-2 S protein antibodies, a collection of significant antibodies are introduced and compared in the fields of the target, source and type.(4) Mechanisms of therapeutic treatments for SARS-CoV-2 varied.Though the antibody and medicine treatments related to the SARS-CoV-2 S protein are of high specificity and great efficacy, the mechanism, safety, applicability and stability of some agents are still unclear and need further assessment.Therefore, to curb the pandemic, researchers in all fields need more cooperation in the development of SARS-CoV-2 antibodies and medicines to face the great challenge.Copyright © Palaeogeography (Chinese Edition).All right reserved.

Inflammatory Pathways Associated with Covid-19 Severity Differ Based on Obesity Status

Background: Severe COVID-19 and obesity are characterized by higher inflammation. We aimed to examine early inflammatory patterns in people with (Ob) and without (NOb) obesity and COVID-19 and how they relate to COVID-19 disease severity Methods: Ob (BMI >30 Kg/m2) and NOb with COVID-19 matched for age, sex and WHO disease severity provided blood early after diagnosis. Immunoassays measured 57 plasma biomarkers reflecting innate immune and endothelial activation, systemic inflammation, coagulation, metabolism and microbial translocation (Fig 1). Between-group differences were assessed by Mann- Whitney. Associations between subsequent maximal COVID-19 severity (mild vs moderate/severe/critical) and biomarkers were explored by logistic regression adjusted for age, sex, hypertension (HTN) and diabetes (DM). Data are median pg/mL [IQR] or n [%] unless stated Results: Of 100 subjects (50 Ob and 50 Nob) presenting between April 2020 and March 2021, characteristics (Ob vs Nob) included: age 65 [23-91] vs 65 [21-95];female sex 27 (48%) vs 28 (56%);BMI 33.7 [30.0-71.8] vs 23.3 [15.3-25.9];disease severity mild 22 [48%] vs 23 [46%], moderate 15 [30%] vs 13 [26%], severe 6 [12%] vs 7 [14%];HTN 30 (60%) vs 17 (34%);DM 19 [38%] vs 6 [12%];days from symptom onset 7 [2-17] vs 8 [1-15];vaccinated 3 (6%) vs 0 (0%). Compared to NOb, Ob had higher IFN-alpha (1.8 [0.6;11] vs 0.9 [0.1;4.7]), CRP (10 mAU/mL [9.6;10.2] vs 9.7 [7.2;10]), IL-1RA (197 [122;399] vs 138 [88;253]), IL-4 (288 AU/mL [161;424] vs 205 [82;333]), vWF (252 [166;383] vs 163 [96;318]), Zonulin (114 ng/mL [77;131] vs 57 [18;106]), Resistin (956 [569;1153] vs 727 [712;1525]), Leptin (3482 [1513;5738] vs 848 [249;2114]), and lower Adiponectin (1.12 mg/L [0.09;1.5] vs 1.5 [1.18;1.93]), all p< 0.05. In both groups higher, proinflammatory IL-18 and lower levels of antiinflammatory CCL22 and IL-5 were associated with higher odds of disease severity, and lower E-selectin with higher disease severity only in Ob. However, in NOb higher type 3 interferons (IL-28A), macrophage activation (sCD163, CCL3) and vascular inflammation markers (ICAM-1, VCAM-1), along with higher S100B, GM-CSF and leptin were also associated with disease severity, a pattern not observed in Ob (Fig 1) Conclusion(s): Although Ob had higher overall levels of inflammation than NOb, few biomarkers predicted subsequent COVID-19 severity in Ob. These differential inflammatory patterns suggest dysregulated immune responses in Ob with COVID-19. (Figure Presented).

Olfactory Performance in Post Mild-to-Moderate Acute Covid-19 across 2 Years

Background: Previous longitudinal studies (n=6) of objective olfaction performance post-acute COVID-19 have a maximum follow-up of 6-month and do not often test biomarkers. Although olfactory dysfunction appears to improve within two months of symptom onset, 4/6 longitudinal studies show persistent olfactory impairment. Method(s): PCR-confirmed COVID-19 patients in the prospective ADAPT cohort (Sydney, Australia) were assessed across 18 acute symptoms and hospitalization status: 40% mild, 50% moderate, 10% severe/hospitalised - none deceased). Blood samples were taken 2 (N=179), 4 (N=148) and 12-month (N=118) post-diagnosis. The NIH Odor Identification Test (OIT) and the Cogstate brief cognitive battery were performed. 58 also had an olfaction test at 24-month. The OIT raw data were transformed into demographically-corrected T-scores. OIT's attrition was completely random and only initial age (40+/-15 versus 47+/-15) differed between patients lost to follow-up and those in the study at 24-month. We tested peripheral neurobiomarkers (NFL, GFAP, S100B, GM-CSF) and immune markers (Interleukin-IL panel: 1-beta, 1Ralpha, 4, 5, 6, 8, 10, 12p40, 12p70, 13, and MCP-1, TNF-alpha and INF-gamma), analyzed as Log transformed and elevated/normal range using published references. Our previous analyses had shown no relationship with the kynurenine pathway, but an association of impaired olfaction and impaired cognition at 2-month only. Linear mixed effect regressions with time effect (months) tested olfaction trajectories (random subject effect) and their association with the biomarkers (main and time interaction). Result(s): At 2 months post-diagnosis 30% had impaired olfaction and those who had acute severe disease were more likely to be impaired (54% versus 26%, p=.009). 21%, 31% and 37% had impaired olfaction at 4, 12 and 24-months. Olfactory performance declined over time (p< .0001), which was dependent on the initial performance (Fig 1). Neurobiomarkers were within the normal range. IFN-gamma, IL-1Ralpha, IL-13 and TNF-alpha increased across time, p< .03-p< .0005. TNF-alpha and IFN-gamma showed a time covariance with poorer olfaction performance. Conclusion(s): Post-acute mild to moderate COVID-19 is associated with a declining olfactory performance up to 2-yr post-diagnosis, especially when initially impaired with the provisio of attrition although random. Olfactory performance decline may be mediated by upregulated immune parameters which are distinct from those driving cognitive changes. (Figure Presented).

Inflammatory and Blood-Brain Barrier Markers after Covid-19 among People with Hiv

Background: COVID-19, the disease caused by SARS-CoV-2, has resulted in devastating morbidity and mortality worldwide. Alarming evidence indicates that long-term adverse outcomes of COVID-19 can affect all major systems of the body, including the immune, respiratory, cardiovascular, and neurological systems. While acute COVID-19 pathology does not appear to be markedly different by HIV status, long-term outcomes of COVID-19 in People with HIV (PWH) are unknown and require further investigation. This study evaluates the inflammatory profile longitudinally up to three months after COVID-19. In addition, markers of the blood-brain barrier (BBB) integrity and vascular dysfunction were also evaluated. Method(s): Plasma samples were collected from 15 males and 6 females with COVID-19 and HIV infection (COVID+/HIV+) and 9 males and 14 females with COVID-19 without HIV infection (COVID+/HIV-) between March 2020 and March 2021. Baseline samples were obtained approx. 10 days after COVID-19 diagnosis (T=0) and three months after (T=3). Mean age group for COVID+/HIV-was 45.4+/-17.8 years for males and 39.7+/-15.3 for females and for COVID+/HIV+ was 52.1+/-12.3 for males and 48.7+/-1 for females (N=15 and 6, respectively). 27 inflammatory molecules were measured by Bio-Plex Multiplex Immunoassay (Bio-Rad) and two markers of BBB and vascular dysfunction (soluble ICAM1 and S100beta) by ELISA. Result(s): Out of 27 inflammatory analytes, 20 had detectable signals. Eotaxin (CCL11) and G-CSF levels were differentially upregulated in the COVID+/HIV+ group as compared to the COVID+/HIV-group in both time point studied (Table 1). IFN-g showed sustained increased levels at T=3 in the COVID+/HIV+ group, whereas there was a significant reduction over time in the COVID+/HIV-group. At T3, inflammatory markers (IL-4, IL-8, IL-13, basic FGF, TNF-alpha, MIP-1alpha, and CCL2) either decreased or remained unchanged in both groups. In contrast, the markers of the BBB disruption and vascular dysfunction, such as S100beta and soluble ICAM-1 increased in the COVID+/HIV+ group, suggesting long-term progressive BBB and vascular alterations. Conclusion(s): HIV-1 may potentiate long COVID-19-induced neuropathology, with progressive BBB breakdown and sustained increase in eotaxin-1 and G-CSF. Plasma inflammatory markers in COVID-19 patients with or without HIV-1 co-infection.

A Case of Bilateral Cerebral Venous Sinus Thrombosis: More Than Meets the Eye.

Cerebral venous sinus thrombosis (CVST) is a rare etiology of stroke that results from inherited and/or acquired conditions, which can present in a variety of symptoms. CVST in the setting of the 2019 coronavirus disease (COVID-19) has rarely been observed. Herein, we present the case of a 32-year-old female with a recent history of COVID-19 subsequently found to have CVST involving bilateral transverse sinuses. Further workup demonstrated several hypercoagulable conditions, which were likely exacerbated by the viral infection. This case demonstrates an atypical outcome for young, COVID-19-positive patients, which emphasizes the importance of diligence when examining symptomatic patients with a history of COVID-19 infection. The patient was treated with apixaban therapy with radiographic resolution of bilateral CVST and improved vision.

Delayed type hypersensitivity (DTH) cutaneous test as a first step to investigate long term immune response to Covid-19 vaccines

Background: As we progress with mass vaccination against SARs-CoV- 2, there are key questions about immunogenicity of Covid vaccines that still are not answered. One of the reasons for this is the lack of suitable cellular immune methods that are easy to perform in large amount of vaccinated individuals. Conventional methods of measuring cellular and humoral immunity are complex and expensive in a pandemic situation. Here we present data about in vivo Delayed Type Hypersensitivity (DTH) results on the long-term follow-up of vaccinated immunocompetent health care workers showing that DTH is a suitable and affordable method to understand immunigenicity elicited by Covid-vaccines. This tool could be used to modify vaccine administration to optimize responses obtained in the vaccinated individuals. Method(s): Forty healthy healthcare workers accepted to participate during the vaccination schedule with a mRNA vaccine against SARS-CoV- 2. We measure the DTH cutaneous response after intradermal test of protein S of SARS-CoV- 2 at day 35 and day 200. At the same time, we perform specific anti-RBD IgG using a classic ELISA before vaccination and on days 35 and 200. Result(s): All forty individuals had a positive DTH skin response al day 35, whereas thirty-nine participants had a positive skin test at day 200 (Figure 1). Moreover, although all 40 individuals showed a humoral response of specific IgG against spike protein at day 35 considered positive, most of them have significant lower levels at day 200. Conclusion(s): DTH responsiveness has been shown to be an ideal and easy method to predict cellular immunity response to mRNA vaccines 200 days after starting immunization schaedule with mRNA vaccine for Covid-19. (Figure Presented).

Protein S Deficiency with Favourable Foetomaternal Outcome: A Case Report

Protein S is a multifunctional plasma protein, whose deficiency, results in a rare congenital thrombophilia, inherited in an autosomal dominant pattern. It can aggravate the hypercoagulable state of pregnancy, when it presents in parallel with the condition, leading to adverse maternal outcomes and foetal loss. A 35-year-old female third gravida having previous 2 deliveries by Lower Segment Caesarean Section (LSCS) presented to emergency at 10 weeks pregnancy with chief complaints of pain and swelling in left thigh since 4-5 days. After thorough investigations and work-up, the patient was diagnosed with Protein S deficiency. She was managed conservatively and was delivered by elective LSCS with bilateral tubal ligation at 38 weeks of gestation with good foetal and maternal outcomes.The rarity of Protein S deficiency along with the successful outcome of the pregnancy makes this a unique case.Copyright © 2023 Journal of Clinical and Diagnostic Research. All rights reserved.

Construction and immunogenicity of recombinant rabies virus expressing S gene of SARS-CoV-2

[Background] The coronavirus disease 2019（COVID-19） pandemic has lasted for nearly three years in the globe, which has not only caused serious harm to humans but also affected companion animals. The COVID-19 vaccines for human have been used globally, while those for animals are rarely reported. [Objective] To develop a bivalent vaccine against both severe acute respiratory syndrome coronavirus 2（SARS-CoV-2） and rabies virus（RABV） for animal use. [Methods] We cloned the S and S1 genes of SARS-CoV-2 into the region between G and L genes of the attenuated RABV vaccine strain rHEP-Flury to construct the recombinant plasmids pHEP-nCOV-S and pHEP-nCOV-S1, respectively.The two plasmids were respectively co-transfected into BHK-21 cells with the helper plasmids and finally the recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 were rescued. The recombinant viruses were confirmed by RT-PCR and direct fluorescent antibody staining against RABV N protein.Western blotting was employed to detect the expression of S and S1 proteins in the cells infected with the recombinant viruses. The growth curves, pathogenicity, and immunogenicity of recombinant viruses were confirmed in NA cells and mice. [Results] The rescued recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 respectively carrying the S and S1 genes of SARS-CoV-2 were confirmed by direct fluorescent antibody assay based on the green fluorescence from the supernatants 7 days post infection.rHEP-nCOV-S1 rather than rHEP-nCOV-S showed stronger proliferation and diffusion abilities than the parental virus rHEP-Flury in NA cells. The specific bands at 72 kDa and 144 kDa in the Western blotting confirmed the efficient expression of S and S1 in the recombinant viruses, respectively. The mice vaccinated with the recombinant viruses did not show significant changes in the body weight compared with those vaccinated with rHEP-Flury, and the recombinant viruses induced the production of neutralizing antibody against RABV in mice. [Conclusion] The production of the recombinant RABV carrying the S/S1 gene of SARS-CoV-2 provides a foundation for the development of the bivalent vaccine against both SARS-CoV-2 and rabies virus for animal use.

Study of Humoral Immunity against Coronavirus Infection COVID-19 in Vaccinated Individuals with Vaccines Available in the Republic of Belarus (Sputnik V (Gam-COVID-Vac), RF and Sinopharm (BBIBP-CorV), PRC)

Relevance. Many countries around the world are developing effective vaccines against SARS-CoV-2. The measure of the effectiveness of the vaccination process has traditionally been antibody production. The frequency and intensity of adverse reactions is also an important factor in making a decision regarding a vaccine. This study presents the results of the evaluation of the formation of humoral immunity and the occurrence of reactions in response to the administration of Sputnik V (Gam-COVID-Vac), RF, and Sinopharm (BBIBP-CorV), PRC. Aim. Analyze immunogenicity and reactogenicity of COVID-19 vaccines used in the Republic of Belarus (Sputnik V and Sinopharm). Materials and methods. Evaluation of postvaccination immune response by enzyme immunoassay and differential enzyme immunoassay for class G immunoglobulins to S-and N-proteins SARS-CoV-2. Blood plasma of the study participants was used as biological material. Blood sampling was performed 3 times: immediately before the first vaccine dose, on day 42, and 6 months after the first vaccine dose. To evaluate the frequency and intensity of postvaccination reactions, study participants were questioned. Results. At 42 days after administration of both vaccines, antibody levels are rising, with a significantly higher quantitative IgG count for the Sputnik V vaccine. This trend is also observed 6 months after the first dose of both vaccines, both among those previously infected with SARS-CoV-2 and those without a history of COVID-19. The comparison of Sputnik V and Sinopharm vaccine groups in terms of IgG (BAU/ml) levels to S-and N-proteins revealed a statistically significant difference in IgG levels to S-protein: the Sputnik V vaccine group had significantly higher IgG levels to S-protein than the Sinopharm vaccine group (p = 0.0000196). The incidence of adverse reactions in this study was 45%. All reactions noted were mild to moderate in severity. The most common were soreness and redness at the injection site, elevated body temperature, and a combination of several reactions. The increased body temperature after vaccination was more common among those vaccinated with the Sputnik V vaccine. Conclusion. Compared to Sinopharm, Sputnik V vaccine produces higher antibody level. Adverse reactions were observed in both vaccinated groups. However, significant statistical differences were found with regard to fever in the Sputnik V vaccine group, which occurred more frequently. © 2023, Numikom. All rights reserved.

The predictive value of coagulation parameters for the course of disease in COVID-19 patients

Introduction COVID-19 is a systemic disease associated with a high incidence of thrombotic complications. In this study we aimed to identify coagulation parameters as predictors of mortality in hospitalized patients with severe COVID- 19 infection. Method We conducted a non-interventional, national, monocentric observational study of patients treated for COVID infection at the ICU at Frankfurt University Hospital. A total of 410 patients were enrolled in the study between April 1, 2020 and December 31, 2021. Patients had to be 18 years or older and the diagnosis was confirmed by COVID real-time PCR. Coagulation parameters were analysed once on admission to the clinic and 5 to 8 days later. Variables studied included thromboplastin time, aPTT, fibrinogen, D-dimers, antithrombin, hs-troponin, all coagulation factors and vWF antigen, protein C and protein S. Data was also collected on age, sex, comorbidities, medication, and invasive ventilation, ECMO therapy and dialysis. In order to compare patients regarding their general disease status, the SAPS-II and the Horovitz index were determined at the beginning and end of the observation period. Univariate and multivariate logistic regression models were then used to screen coagulation parameters for association with mortality in critically ill COVID patients. Results The arithmetic mean age of patients was 60.9 ( +/- 14.7) years, with 76.1 % being male. Of 410 patients, 259 (63.2 %) received invasive ventilation, 95 (23.2 %) received ECMO therapy and 105 (25.6 %) received renal replacement therapy. The median inpatient length of stay was 16 (IQR: 10-29) days and ICU length of stay was 12 (IQR: 6-25) days. 176 patients (43 %) died because of their COVID disease, 234 (57 %) were discharged home or to other facilities for further treatment. In univariate logistic regression, increased age (OR = 1,029, 95 %-CI [1,013- 1,1,044]), higher SAPS-II (OR = 1,031, 95 %-CI [1,018-1,045]), fibrinogen (OR = 1,002, 95 %-CI [1,001-1,003]), FVIII (OR = 1,004, 95 %-CI [1,001-1,007]) and vWF antigen (OR = 1,005, 95 %-CI [1,003-1,007]) as well as lower antithrombin (OR = 0,981, 95 %-CI [0,971-0,991]), FII (OR = 0,983, 95 %-CI [0,972-0,993]), FXIII (OR = 0,992, 95 %-CI [0,986-0,999]), Horovitz index at admission (OR = 0,994, 95 %-CI [0,990-0,997]) and decreased protein C activity (OR = 0,989, 95 %-CI [0,982-0,996]) were associated with increased mortality. In the final multivariate regression analysis with backward elimination, low antithrombin activity (OR = 0.987, 95 %-CI [0.974-1.000]), high vWF antigen levels (OR = 1.004, 95 %-CI [1.002-1.007]) and a low Horovitz index (OR = 0.993, 95 %-CI [0.989-0.997]) were identified as independent predictive factors for increased mortality. Conclusion In the study of 410 COVID patients requiring intensive care, the Horovitz index, antithrombin activity and vWF antigen on hospital admission were identified as independent predictors of mortality.

A Persistent Nightmare: A Case of Recurrent Brachial Artery Thrombosis

Background Brachial artery thrombosis can be seen with thromboembolism, hypercoagulability, and arterial thoracic outlet syndrome. Case A 33-year-old healthy female construction worker presented with right hand discoloration and pain. She suffered a COVID-19 infection 8 weeks prior with hand symptoms developing shortly thereafter. She could no longer work due to the pain. Duplex ultrasound and CTA of the right upper extremity (Figure) demonstrated localized thrombosis of the right brachial artery. The workup yielded no aortic or intracardiac thrombus, and cardiac event monitor showed no atrial arrhythmia. She underwent thrombectomy with brachial artery stenting and was found, during surgery, to have distal ulnar artery occlusion. Two days post-op, she had recurrent pain and was found to have brachial artery recurrent thrombosis. She underwent urgent brachial-brachial bypass. Arm pain continued despite graft patency, so ulnarpalmar bypass was performed. Decision-making Hypercoagulability workup, including antiphospholipid antibody, protein C, protein S, homocysteine, and Lp(a), was negative. Neither central thrombus on TEE nor evidence of thoracic outlet syndrome was found. As a diagnosis of exclusion, brachial artery thrombosis was ascribed to COVID infection. Despite rivaroxaban, the patient developed gangrene (Panel C) requiring partial digit amputation. Conclusion We present a case of COVID-19-induced recurrent brachial artery thrombosis despite surgical intervention. [Formula presented]Copyright © 2023 American College of Cardiology Foundation

Evaluation of protein C and S levels in patients with COVID-19 infection and their relation to disease severity.

Background: The COVID-19 pandemic has been associated with millions of deaths around the world. One of the important causes of death associated with COVID-19 was pulmonary thromboembolism. The risk for venous thromboembolism was markedly increased in patients with COVID-19 especially those admitted to the intensive care unit. The aims of our study were to measure the protein C and S levels in COVID-19-infected patients in comparison with the normal population and to assess the correlation of protein C and S levels in the plasma to the severity of infection. Methods: This was a case-control study measuring the protein C and S levels in patients infected with COVID-19 at the time of diagnosis compared to the normal population. The study included one hundred participants, sixty of them are patients with COVID-19, and forty of them are normal healthy adults. The patient group was subclassified into three subgroups according to disease severity: mild, moderate, and severe COVID-19 infections. Results: The activity of protein C in the patient group serum was significantly lower than that in the control group serum (79.35 ± 26.017 vs 97.43 ± 15.007; p < 0.001). Protein S is also significantly decreased in patients' serum when compared to the control group (70.233 ± 22.476 vs 91 ± 14.498; p < 0.001). There was a statistically significant decrease in the levels of protein C and S associated with the increase in disease severity (p < 0.05). However, protein S showed no statistically significant difference between the moderate and severe disease subgroups. Conclusion: The study concluded that the levels of protein C and S activities were both decreased in patients with COVID-19 when compared to the healthy population. It also concluded that the decrease in their levels is statistically significant in relation to the disease severity.

Dysregulation of Protein S in COVID-19.

Coronavirus Disease 2019 (COVID-19) has been widely associated with increased thrombotic risk, with many different proposed mechanisms. One such mechanism is acquired deficiency of protein S (PS), a plasma protein that regulates coagulation and inflammatory processes, including complement activation and efferocytosis. Acquired PS deficiency is common in patients with severe viral infections and has been reported in multiple studies of COVID-19. This deficiency may be caused by consumption, degradation, or clearance of the protein, by decreased synthesis, or by binding of PS to other plasma proteins, which block its anticoagulant activity. Here, we review the functions of PS, the evidence of acquired PS deficiency in COVID-19 patients, the potential mechanisms of PS deficiency, and the evidence that those mechanisms may be occurring in COVID-19.

Increased risk of dialysis circuit clotting in hemodialysis patients with COVID-19 is associated with elevated FVIII, fibrinogen and D-dimers.

INTRODUCTION: Severe COVID-19 infections increase the risk of thrombotic events and Intensive Care Units reported increased extracorporeal circuit clotting (ECC) in COVID-19 patients with acute kidney injury. We wished to determine whether hemodialysis (HD) patients with COVID-19 also have increased risk of circuit clotting. METHODS: We reviewed coagulation studies and HD records, 4 weeks before and after COVID-19 polymerase chain reaction detection in HD patients between April 2020 and June 2021. FINDINGS: Sixty-eight (33.5%) of 203 HD patients with COVID-19, 65% male, mean age 64.9 ± 15.3 years, experienced some circuit clotting, and no clotting recorded prior to positive test results. In those who experienced ECC, prothrombin, activated partial thromboplastin or thrombin times were not different, whereas median factor VIII (273 [168-419] vs. 166 [139-225] IU/dl, p < 0.001), D-dimers (2654 [1381-6019] vs. 1351 [786-2334] ng/ml, p < 0.05), and fibrinogen (5.6 ± 1.4 vs. 4.9 ± 1.4 g/L, p < 0.05) were greater. Antithrombin (94 [83-112] vs. 89 [84-103] IU/dl), protein C (102 [80-130] vs. 86 [76-106] IU/dl), protein S (65 [61-75] vs. 65 [52-79] IU/dl) and platelet counts (193 [138-243] vs. 174 [138-229] × 109 /L) did not differ. On multivariable logistic analysis, circuit clotting was associated with log factor VIII (odds ratio [OR] 14.8 (95% confidence limits [95% CL] 1.12-19.6), p = 0.041), fibrinogen (OR 1.57 [95% CL 1.14-21.7], p = 0.006) and log D dimer (OR 4.8 [95% CL 1.16-12.5], p = 0.028). DISCUSSION: Extracorporeal circuit clotting was increased within 4 weeks of testing positive for COVID-19. Clotting was associated with increased factor VIII, fibrinogen and D-dimer, suggesting that the risk of circuit clotting was related to the inflammatory response to COVID-19.

Evidence for multiple binding modes in the initial contact between SARS-CoV-2 spike S1 protein and cell surface glycans.

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the 'open' conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required.

Decreased protein C activity, lower ADAMTS13 antigen and free protein S levels accompanied by unchanged thrombin generation potential in hospitalized COVID-19 patients.

INTRODUCTION: COVID-19 is associated with an increased thromboembolic risk. However, the mechanisms triggering clot formation in those patients remain unknown. PATIENTS AND METHODS: In 118 adult Caucasian severe but non-critically ill COVID-19 patients (median age 58 years; 73 % men) and 46 controls, we analyzed in vitro plasma thrombin generation profile (calibrated automated thrombogram [CAT assay]) and investigated thrombophilia-related factors, such as protein C and antithrombin activity, free protein S level, presence of antiphospholipid antibodies and factor V Leiden R506Q and prothrombin G20210A mutations. We also measured circulating von Willebrand factor (vWF) antigen and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) antigen and activity. In patients, blood samples were collected on admission to the hospital before starting any therapy, including heparin. Finally, we examined the relationship between observed alterations and disease follow-up, such as thromboembolic complications. RESULTS: COVID-19 patients showed 17 % lower protein C activity, 22 % decreased free protein S levels, and a higher prevalence of positive results for IgM anticardiolipin antibodies. They also had 151 % increased vWF, and 27 % decreased ADAMTS13 antigens compared with controls (p < 0.001, all). On the contrary, thrombin generation potential was similar to controls. In the follow-up, pulmonary embolism (PE) occurred in thirteen (11 %) patients. They were characterized by a 55 % elevated D-dimer (p = 0.04) and 2.7-fold higher troponin I (p = 0.002) during hospitalization and 29 % shorter time to thrombin peak in CAT assay (p = 0.009) compared to patients without PE. CONCLUSIONS: In COVID-19, we documented prothrombotic abnormalities of peripheral blood. PE was characterized by more dynamic thrombin generation growth in CAT assay performed on admittance to the hospital.

Epitope-directed anti-SARS-CoV-2 scFv engineered against the key spike protein region could block membrane fusion.

The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively.

Is Undected Inherited Thrombophilia The Hades Of COVID-19?

Introduction: Thrombophilia is a co-morbidity in the general population with a prevalence of 5%. However, it is not known whether it affects severity of COVID-19. It may be possible that undiagnosed thrombophilia exaggerates an already prothrombotic state in COVID- 19 patients and hence, results in severe disease. Aims & Objectives: To study the association of underlying thrombophilia with severity of COVID-19 in post COVID patients after a minimum of 6 weeks of recovery. Material(s) and Method(s): Eighty RT-PCR confirmed adult covid patients (40 severe,40 non-severe) post 6 weeks of recovery were included. The venous blood in EDTA and citrate vials was tested for complete blood counts and coagulation parameters such as Prothrombin time, activated partial prothrombin time, Lupus anticoagulant by dRVVT, Antithrombin-III, Protein C and S). Result(s): 6/40 patients had Protein C deficiency in severe category and none in non severe. 7/40 patients had Protein S deficiency in severe category and 1 patient was deficiant in non-severe group. Conclusion(s): Thrombophilia was detected significantly in patients with severe COVID even after 6 weeks of recovery, indicating that undetected underlying thrombophilia could be a factor affecting disease severity. The common abnormalities detected were Protein C & S deficiency. This is a novel finding which needs to be explored further with extensive thrombophilia profiles. The influence of underlying thrombophilia in patients who succumbed to the disease is not known and cannot be known retrospectively. However, the extrapolation of this study suggests that thrombophilia may be an important influence on severity and mortality. (Table Presented).

Graphene Oxide Decreases Pro-Inflammatory Proteins Production in Skeletal Muscle Cells Exposed to SARS-CoV-2 Spike Protein.

Aim: The experiments aimed to document the presence of the ACE2 receptor on human muscle cells and the effects of the interaction of these cells with the spike protein of the SARS-CoV-2 virus in terms of induction of pro-inflammatory proteins, as well as to assess the possibility of reducing the pool of these proteins with the use of graphene oxide (GO) flakes. Methods: Human Skeletal Myoblast (HSkM), purchased from Gibco were maintained in standard condition according to the manufacturer's instruction. The cells were divided into 4 groups; 1. C-control, 2. S-with addition of spike protein, 3. GO-with the addition of graphene oxide, 4. GO-S-with addition of GO followed by the addition of S protein. Protein S (PX-COV-P049) was purchased from ProteoGenix (France). GO was obtained from Advanced Graphene Products (Zielona Gora, Poland). The influence of all the factors on the morphology of cells was investigated using light and confocal microscopy. ACE2 protein expression on muscle cells was visualized and 40 pro-inflammatory cytokines were investigated using the membrane antibody array method. The protein profile of the lysate of cells from individual groups was also analyzed by mass spectrometry. Conclusion: The experiments confirmed the presence of the ACE2 receptor in human skeletal muscle cells. It has also been documented that the SARS-CoV-2 virus spike protein influences the activation of selected pro-inflammatory proteins that promote cytokine storm and oxidative stress in muscle cells. The use of low levels of graphene oxide does not adversely affect muscle cells, reducing the levels of most proteins, including pro-inflammatory proteins. It can be assumed that GO may support anti-inflammatory therapy in muscles by scavenging proteins that activate cytokine storm.

Safety and Immunogenicity of a Single Dose of BNT162b2 COVID-19 mRNA Vaccine in a Warfarin-Treated Protein S Deficient Patient: A Case Report and Literature Review.

Patients with protein S (PS) deficiency possibly have a higher risk of developing severe COVID-19 disease. Therefore, vaccination against SARS-CoV-2 infections is recommended for PS-deficient patients. However, there are limited data regarding the safety and immunogenicity of the currently available COVID-19 mRNA vaccine in PS-deficient patients. We report a case of monitoring the antibody response of a 40-year-old female diagnosed with PS deficiency and on warfarin treatment following a single dose of BNT162b2 mRNA vaccine. Antibody against the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (anti-S) was measured on days 7, 14, and 21 after vaccination. Seroconversion was detected on day 21 but was possibly lower than the anti-S level previously reported in healthy individuals after receiving the first dose of the BNT162b2 mRNA vaccine. There were no local and systemic events reported up to 7 days in this patient after vaccination. This case highlights that the administration of the BNT162b2 vaccine had a favourable safety profile, and the second dose of the vaccine is required to provide the optimal protection against SARS-CoV-2 infection in PS-deficient patients.