Immunogenicity evaluation of Gam-COVID-Vac (Sputnik V)

In November 2020, the Armed Forces of the Russian Federation began mass immunisation of the personnel with Gam-COVID-Vac (Sputnik V), the first Russia vaccine against the new coronavirus infection (COVID-19). Thus, it became necessary to assess post-vaccination antibody levels and the duration and intensity of humoral immunity to COVID-19. The aim of the study was to investigate the immunogenicity and efficacy of Gam-COVID-Vac in military medical staff after vaccination. Materials and methods: the authors determined the presence of specific antibodies in the serum of individuals immunised with Gam-COVID-Vac (477 volunteers) and COVID-19 convalescents (73 patients), using virus neutralisation (VN), enzyme-linked immunosorbent assay (ELISA) with reagent kits by several manufacturers, and immunoblotting. The results of the study were evaluated using analysis of variance. Results: VN detected virus neutralising antibodies in 90.7% of vaccinated subjects;ELISA, in 95.4%. Both VN and ELISA showed lower antibody levels in the vaccinated over 50 years of age. ELISA demonstrated a significantly higher concentration of anti-SARS-CoV-2 spike IgG in the Gam-COVID-Vac group than in the COVID-19 convalescent group. The correlation between antibody detection results by VN and ELISA was the strongest when the authors used their experimental reagent kit for quantitative detection of virus neutralising antibodies by competitive ELISA with the recombinant human ACE2 receptor. Having analysed the time course of neutralising antibody titres, the authors noted a significant, more than two-fold decrease in geometric means of the titres three months after administration of the second vaccine component. Conclusions: the subjects vaccinated with Gam-COVID-Vac gain effective humoral immunity to COVID-19. The decrease in titres indicates the need for revaccination in 6 months.

Influence of the COVID-19 Pandemic on the Prevalence Pattern of Allergens.

INTRODUCTION: The effect of the COVID-19 pandemic on allergic diseases is not certain, as people's living habits and the environment have been affected by the pandemic. The present study described the influence of the COVID-19 pandemic on the allergen sensitization rate in patients with allergic diseases in central China. The results provide reliable epidemiological data for the prevention and control of allergic diseases during the COVID-19 epidemic. METHODS: Data were collected from a total of 6,915 patients with symptoms of allergic diseases who visited the Third Xiangya Hospital of Central South University in China for allergen testing from January 1, 2018, to December 31, 2021. Patients were divided into a children group (<14 years old), youth group (15∼44 years old), middle-aged group (45∼59 years old), and elderly group (>60 years old). Immunoblotting was used to detect 20 serum allergen-specific IgE (sIgE) antibodies in patient serum samples. We compared the positive rates of various allergens in different age and sex groups before and during the COVID-19 epidemic, and the prevalence data of sIgE sensitization were analysed. RESULTS: Among the 6,915 patients with symptoms of allergic diseases, 2,838 (41.04%) patients were positive for at least one of the allergens. The top three positive rates of inhaled allergens were Dermatophagoides farinae (1,764 cases, 25.51%), Dermatophagoides pteronyssinus (1,616 cases, 23.37%), and house dust (645 cases, 9.33%). The top three positive rates of food allergens were eggs (686 cases, 9.92%), milk (509 cases, 7.36%), and crabs (192 cases, 2.78%). The total positive rate of allergens was higher in men (46.99%) than in women (37.30%). Compared to 2 years before the COVID-19 epidemic, the rate of sensitization to indoor inhalant allergens increased, but outdoor inhalant allergens showed no significant change. The positive rates of milk and eggs peaked during the outbreak of COVID-19 (2020) then declined in 2021. The total positive rate of allergens was higher in males than females before and during the COVID-19 epidemic, but more allergens were different between males and females during the pandemic. Compared to middle-aged and older adults, the children and youth groups were more susceptible to allergic diseases, and they exhibited an increasing positive rate for most common allergens, especially indoor inhalant allergens, during the COVID-19 epidemic than before the pandemic. CONCLUSION: D. pteronyssinus and D. farinae are the most common allergens in South China. Under the background of normalization of epidemic prevention, indoor inhaled allergens should be first in the prevention and control of allergic diseases, and a combination of various indoor cleaning measures should be used to improve the efficiency of interventions.

RNA INTERFERENCE IS THE BASIS OF HUMAN ANTIVIRAL DEFENSE.

Comparative analysis of antiviral protective mechanisms in protozoa and RNA interference of multicellular organisms has revealed their similarity, also providing a clue to understanding the adaptive immunity. In this article, we present the latest evidence on the importance of RNA-guided gene regulation in human antiviral defense. The role of neutralizing antibodies and interferon system in viral invasion is considered. The new concept has been introduced, i.e., antiviral protection of any living organism is based on the intracellular RNA-guided mechanisms. Simple and effective defense against viruses is that spacer segment of the viral DNA is inserted into the cellular chromosomes. Upon re-infection, the RNA transcript of the spacer directs nuclease enzymes against the foreign genome. This is a really adaptive immune defense that any cell potentially possesses. In humans, the interferon system provides an additional tool for early suppression of viral infections which shifts the cells to the alert regimen, thus preventing further spread of infection. The main task of the human central immune system is to maintain integrity and combat foreign organisms. Accordingly, a suitable index of acquired antiviral immunity should be a presence of specific spacer markers in DNA samples from reconvalescent persons, rather than detection of neutralizing antibodies, B and T memory cells. This article is addressed primarily to general medical community, and its practical conclusions are as follows: 1. Presence or absence of specific antibodies to SARS-CoV-2 is not a prognostic sign of the disease. Detection of specific antibodies in blood simply reflects the fact that the person has contacted with the viral agent. Absence of antibodies does not mean a lack of such contact, and the persons with high titers of specific antibodies are not protected from re-infection with SARS-CoV-2. 2. PCR testing: The PCR results may remain "false positive" in those subjects who have had COVID-19, if the genetic material is taken from the site of initial virus contraction (mainly, nasopharynx). In our opinion, negative PCR tests for COVID-19 in blood plasma and urine will be a more correct index for the absence of the disease, even with positive PCR tests from the nasopharyngeal samples. 3. It is necessary to draw attention of general practitioners to potential usage of retinol in prevention and treatment of COVID-19, given the importance of RLR receptors in recognition of viral RNAs and positive experience of vitamin A administration in measles, another dangerous viral disease. Copyright © 2022, SPb RAACI.

RNA INTERFERENCE IS THE BASIS OF HUMAN ANTIVIRAL DEFENSE

Comparative analysis of antiviral protective mechanisms in protozoa and RNA interference of multicellular organisms has revealed their similarity, also providing a clue to understanding the adaptive immunity. In this article, we present the latest evidence on the importance of RNA-guided gene regulation in human antiviral defense. The role of neutralizing antibodies and interferon system in viral invasion is considered. The new concept has been introduced, i.e., antiviral protection of any living organism is based on the intracellular RNA-guided mechanisms. Simple and effective defense against viruses is that spacer segment of the viral DNA is inserted into the cellular chromosomes. Upon re-infection, the RNA transcript of the spacer directs nuclease enzymes against the foreign genome. This is a really adaptive immune defense that any cell potentially possesses. In humans, the interferon system provides an additional tool for early suppression of viral infections which shifts the cells to the alert regimen, thus preventing further spread of infection. The main task of the human central immune system is to maintain integrity and combat foreign organisms. Accordingly, a suitable index of acquired antiviral immunity should be a presence of specific spacer markers in DNA samples from reconvalescent persons, rather than detection of neutralizing antibodies, B and T memory cells. This article is addressed primarily to general medical community, and its practical conclusions are as follows: 1. Presence or absence of specific antibodies to SARS-CoV-2 is not a prognostic sign of the disease. Detection of specific antibodies in blood simply reflects the fact that the person has contacted with the viral agent. Absence of antibodies does not mean a lack of such contact, and the persons with high titers of specific antibodies are not protected from re-infection with SARS-CoV-2. 2. PCR testing: The PCR results may remain "false positive” in those subjects who have had COVID-19, if the genetic material is taken from the site of initial virus contraction (mainly, nasopharynx). In our opinion, negative PCR tests for COVID-19 in blood plasma and urine will be a more correct index for the absence of the disease, even with positive PCR tests from the nasopharyngeal samples. 3. It is necessary to draw attention of general practitioners to potential usage of retinol in prevention and treatment of COVID-19, given the importance of RLR receptors in recognition of viral RNAs and positive experience of vitamin A administration in measles, another dangerous viral disease. © 2022, SPb RAACI.

Ensovibep antiviral activity in ambulatory patients with COVID-19 is independent of baseline anti-SARS-CoV-2 antibodies and exhibits minimal selective pressure - Results from the placebo-controlled EMPATHY trial

Background. Ensovibep is a multi-specific DARPin (designed ankyrin repeat protein) antiviral in clinical development for treatment of COVID-19. In the Phase 2 EMPATHY study, ensovibep demonstrated greater viral load decline versus placebo. Here we report (1) the efficacy of ensovibep in patients with and without anti-SARS-CoV-2 antibodies at baseline and (2) SARS-CoV-2 mutation emergence data with treatment. Methods. Eligible ambulatory patients with >=2 COVID-19 symptoms (onset within 7 days) and positive SARS-CoV-2 rapid antigen test on day of dosing, were randomized (1:1:1:1) to ensovibep (600, 225 or 75 mg) or placebo as single, IV infusion. Chemiluminescent immunoassays were used for antibody detection (SARS-CoV-2 S1/S2 IgG and SARS-CoV-2 IgM). A pre-specified subgroup analysis was performed based on baseline anti-SARS-CoV-2 antibody status. Analysis of changes in viral genome from baseline to post baseline was performed to evaluate treatment-emergent mutations. Results. Of the patients analyzed, 48.5% had anti-SARS-CoV-2 antibodies at baseline. Baseline log10 SARS-CoV-2 viral load (mean +/-SD) was similar across groups [ensovibep (all doses) 6.5 +/-1.5, placebo 6.2 +/-1.5];> 90% were infected with the Delta (B.1.617.2) variant. SARS-CoV-2 viral load reduction up to Day 8 showed similar effects in favor of ensovibep compared with placebo regardless of the presence of anti-SARS-CoV-2 antibodies (Figure 1). Patients in ensovibep 75 mg, 600 mg, and placebo groups had comparable incidences of emergent mutations, with a higher incidence in the 225 mg group. Based on analysis of 70% of the expected viral sequencing data, two mutations in the key binding residues of ensovibep were observed (Y489H and F486L) in a total of three patients treated with ensovibep. These patients either cleared virus by Day 8 or mutations were transient (occurred at a single time point but not later in the course of infection). (Figure Presented) Conclusion. Ensovibep effectively reduces SARS-CoV-2 viral load regardless of the presence of anti-SARS-CoV-2 antibodies at baseline. Furthermore, there were no emerging mutations of concern, indicating that a single dose administration of ensovibep is associated with minimal selective pressure.

Cerebral Venous and Sinus Thrombosis with Immunological Pathogenesis: VITT and Pre-VITT

In this review, we summarise the current knowledge on vaccine-induced immune thrombotic thrombocytopenia (VITT) and new insights into its underlying pathogenesis. VITT is characterised by severe thromboses occurring 5-20 days after vaccination with an adenoviral vector-based SARS-CoV-2 vaccine (AstraZeneca or Johnson & Johnson). Thromboses typically involve the cerebral sinus and venous system. Routine laboratory analyses show thrombocytopenia and high D-dimer levels. The pathogenesis is based on immunological processes similar to those in heparin-induced thrombocytopenia. Accordingly, VITT is associated with high-titre immunoglobulin G directed against platelet factor 4 (PF4). Interaction with adenoviral vector-based vaccines leads to modifications of PF4 allowing antibody-producing cells to identify PF4. Anti-PF4 antibodies activate platelets through FcgammaIIa receptors. The detection of platelet-activating anti-PF4 antibodies confirms the diagnosis of VITT. Treatment is based on anticoagulation, which inhibits thrombin itself or thrombin formation, and high-dose intravenous immunoglobulin G, which inhibits cell activation via FcgammaIIa receptors. In severe cases, plasma exchange could also be an option. In some patients, a pre-VITT syndrome precedes VITT. Pre-VITT patients typically present with severe headache before thromboses are manifest. The early identification of a pre-VITT syndrome allows for the prevention of thrombotic complications. The specific dynamics of the immune reaction in VITT correspond to a transient, secondary immune response. Current studies address how PF4 binds to different adenoviral proteins and investigate the functional role of other vaccine components. Some of these factors contribute to the induction of a pro-inflammatory danger signal that triggers the first stage of VITT pathogenesis. In the second stage, high-titre anti-PF4 antibodies activate platelets and granulocytes. In a process called NETosis (neutrophil extracellular traps), activated granulocytes release DNA. Anti-PF4 antibodies then bind to complexes of PF4 and DNA. This enhances further cell activation via Fcgamma receptors and consequently also the formation of thrombin. At the end of the article, we comment on how the current knowledge on VITT may influence global vaccination campaigns against SARS-CoV-2 and we address how anti-PF4 antibodies may be involved in recurrent arterial and venous thromboses not associated with VITT and HIT. Copyright © 2022 Georg Thieme Verlag. All rights reserved.

Antibody-Capture Enzyme-Linked Immunosorbent Assay for Detection of Antibody to Turkey Coronavirus Using Infectious Bronchitis Virus or Recombinant Nucleocapsid Protein as Coating Antigen

Turkey coronavirus (TCoV) infection continues to threaten turkey industry. Because specific treatment and effective vaccination program are not available, rapid and cost-effective detection of antibodies to TCoV infection is an important control measure to monitor the disease status in the fields. Two antibody-capture enzyme-linked immunosorbent assay (ELISA) procedures for detection of antibodies to TCoV are outlined in this chapter. One ELISA method uses chicken infectious bronchitis coronavirus (IBV) as the coating antigen based on antigenic cross-reactivity between TCoV and IBV. The other method relies on a recombinant TCoV nucleocapsid protein. Both methods are useful for serological diagnosis of TCoV infection in the turkey flocks. Copyright © Springer Science+Business Media New York 2016.

Development of fluorescent lateral flow immunoassay for SARS-CoV-2-specific IgM and IgG based on aggregation-induced emission carbon dots.

Understanding the dynamic changes in antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for evaluating the effectiveness of the vaccine and the stage for the recovery of the COVID-19 disease. A rapid and accurate method for the detection of SARS-CoV-2-specific antibodies is still urgently needed. Here, we developed a novel fluorescent lateral flow immunoassay (LFA) platform for the detection of SARS-CoV-2-specific IgM and IgG by the aggregation-induced emission carbon dots conjugated with the SARS-CoV-2 spike protein (SSP). The aggregation-induced emission carbon dots (AIE-CDs) are one of the best prospect fluorescent probe materials for exhibiting high emission efficiency in both aggregate and solid states. The AIE-CDs were synthesized and displayed dual fluorescence emission, which provides a new perspective for the design of a high sensitivity testing system. In this work, the novel LFA platform adopted the AIE carbon dots, which are used to detect SARS-CoV-2-specific IgM and IgG conveniently. Furthermore, this sensor had a low LOD of 100 pg/ml. Therefore, this newly developed strategy has potential applications in the areas of public health for the advancement of clinical research.

Automated, flow-based chemiluminescence microarray immunoassay for the rapid multiplex detection of IgG antibodies to SARS-CoV-2 in human serum and plasma (CoVRapid CL-MIA).

In the face of the COVID-19 pandemic, the need for rapid serological tests that allow multiplexing emerged, as antibody seropositivity can instruct about individual immunity after an infection with SARS-CoV-2 or after vaccination. As many commercial antibody tests are either time-consuming or tend to produce false negative or false positive results when only one antigen is considered, we developed an automated, flow-based chemiluminescence microarray immunoassay (CL-MIA) that allows for the detection of IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD), spike protein (S1 fragment), and nucleocapsid protein (N) in human serum and plasma in less than 8 min. The CoVRapid CL-MIA was tested with a set of 65 SARS-CoV-2 serology positive or negative samples, resulting in 100% diagnostic specificity and 100% diagnostic sensitivity, thus even outcompeting commercial tests run on the same sample set. Additionally, the prospect of future quantitative assessments (i.e., quantifying the level of antibodies) was demonstrated. Due to the fully automated process, the test can easily be operated in hospitals, medical practices, or vaccination centers, offering a valuable tool for COVID-19 serosurveillance. Graphical abstract.

Evaluating the Value of Anti-SARS-CoV-2 Antibody-Based Tests for COVID-19 Diagnosis.

BACKGROUND: The early detection of COVID-19 patients is fundamental for containing the pandemic. A reverse-transcriptase quantitative polymerase chain reaction (RT-PCR), which detects SARS-CoV-2 RNA, is the gold standard diagnostic test, although it can contribute to false-negative results. Consequently, supplementary diagnostic tests are urgently needed. METHODS: To assess the value of anti-SARS-CoV-2 antibody-based tests for confirming COVID-19, a retrospective study was conducted on 3120 inbound overseas travelers who underwent a 14-day government quarantine in Xiamen from August 2020 to October 2020. The diagnostic accuracy of the total antibody that detected the anti-SARS-CoV-2 antibody and the RT-PCR that detected SARS-CoV-2 RNA was determined in comparison to the clinical diagnosis. RESULTS: The COVID-19 positive rate was 3.14% (98/3120). The sensitivity and specificity of the RT-PCR test on the first day of quarantine were 14.29% and 100%, respectively, and the sensitivity and specificity of the total antibody were 93.88% and 99.40%, respectively. The kappa value between an RT-PCR on the first day of quarantine and a clinical diagnosis was 0.24 (95% CI, 0.14-0.35), indicating poor consistency. The kappa value between total antibodies and a clinical diagnosis was 0.88 (95% CI, 0.83-0.93), indicating perfect consistency. There were no differences in the positive rates of an RT-PCR in symptomatic COVID-19 (7.41% (2/27)) and asymptomatic COVID-19 (16.90 (12/71) (p = 0.338). Similarly, the positive rate of the total antibody tests showed no difference in symptomatic COVID-19 (96.30% (26/27)) and asymptomatic COVID-19 (92.96% (66/71)) (p = 0.676). CONCLUSION: SARS-CoV-2 antibodies are developed by the body in response to an infection or after vaccination; this can easily lead to a missed diagnosis. In the context of low sensitivity for an RT-PCR, SARS-CoV-2 antibody detection is an effective adjunct to RT-PCR detection, which can improve the diagnostic accuracy of COVID-19 and provide an effective complement to the false-negative results of an RT-PCR.

Accuracy of the hyphen Zymutest IgG ELISA assay for anti-platelet factor 4 antibody detection after ChAdOx1 nCov-19 vaccination in diagnosing vaccine-induced immune thrombotic thrombocytopenia (VITT)

Background: Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT) is a rare, anti-platelet factor 4 (PF4) antibody-mediated prothrombotic syndrome associated with the AstraZeneca (AZ) Covid-19 vaccination. Diagnostic criteria include thrombosis, thrombocytopenia, appropriate timing post AZ vaccine, and demonstration of an anti-PF4 antibody by enzyme-linked (Table Presented) immunosorbent assay (ELISA). ELISA methods have varying sensitivity and specificity for detecting anti-PF4 antibodies, therefore, functional assays (modified serotonin release and flow cytometry) assist in diagnosis. In Australia, the primary assay for detection of anti-PF4 antibodies is the Asserachrom HPIA IgG ELISA. Aim(s): To assess the accuracy of an alternate ELISA method for suspected VITT compared to the gold standard (clinicopathological diagnosis). Method(s): 96 stored frozen samples from patients with suspected VITT, previously analysed by Asserachrom HPIA IgG ELISA (Stago, Melbourne, Australia), were tested using the Hyphen Biomed Zymutest HIA IgG ELISA (Haematex Research, Sydney, Australia). These patients had confirmed thrombosis within 42 days post first dose of AZ vaccine. Cases were classified as 'serologically confirmed VITT' if clinicopathological criteria were met, and initial ELISA testing and/or functional assays were positive. Results were interpreted as positive or negative for anti-PF4 antibodies using the manufacturer's cut-offs derived for the diagnosis of Heparin-Induced Thrombocytopenia. Result(s): 96 patient samples were tested, including 35 classified as VITT (Table 1). The Hyphen assay had a moderate sensitivity (82%), including 2 probable false negatives on initial testing which were strongly positive by Hyphen assay. The specificity was 93%, including 5 probable false positives on initial ELISA which were negative by Hyphen assay (Table 2). Conclusion(s): The Hyphen ELISA is suitably accurate for the diagnosis of VITT. These data highlight the limitations of using a single ELISA testing method for anti-PF4 antibody detection. In cases with high clinical suspicion, a second ELISA method or functional assay should be considered.

Thickness-Sensing Sandwiched Plasmonic Biosensors Enable Label-Free Naked-Eye Antibody Quantification.

Clinical serology assays for detecting the antibodies of the virus are time-consuming, are less sensitive/selective, or rely on sophisticated detection instruments. Here, we develop a sandwiched plasmonic biosensor (SPB) for supersensitive thickness-sensing via utilizing the distance-dependent electromagnetic coupling in sandwiched plasmonic nanostructures. SPBs quantitatively amplify the thickness changes on the nanoscale range (sensitivity: ∼2% nm-1) into macroscopically visible signals, thereby enabling the rapid, label-free, and naked-eye detection of targeted biomolecular species (via the thickness change caused by immunobinding events). As a proof of concept, this assay affords a broad dynamic range (7 orders of magnitude) and a low LOD (∼0.3 pM), allowing for the extremely accurate SARS-CoV-2 antibody quantification (sensitivity/specificity: 100%/∼99%, with a portable optical fiber device). This strategy is suitable for high-throughput multiplexed detection and smartphone-based sensing at the point-of-care, which can be expanded for various sensing applications beyond the fields of viral infections and vaccination.

Detection of SARS-CoV-2 antibodies in serum and dried blood spot samples of vaccinated individuals using a sensitive homogeneous proximity extension assay.

A homogeneous PCR-based assay for sensitive and specific detection of antibodies in serum or dried blood spots (DBS) is presented and the method is used to monitor individuals infected with or vaccinated against SARS-CoV-2. Detection probes were prepared by conjugating the recombinant spike protein subunit 1 (S1), containing the receptor binding domain (RBD) of SARS-CoV-2, to each of a pair of specific oligonucleotides. The same was done for the nucleocapsid protein (NP). Upon incubation with serum or DBS samples, the bi- or multivalency of the antibodies (IgG, IgA or IgM) brings pairs of viral proteins with their conjugated oligonucleotides in proximity, allowing the antibodies to be detected by a modified proximity extension assay (PEA). Anti-S1 and anti-NP antibodies could be detected simultaneously from one incubation reaction. This Antibody PEA (AbPEA) test uses only 1 µl of neat or up to 100,000-fold diluted serum or one ø1.2 mm disc cut from a DBS. All 100 investigated sera and 21 DBS collected prior to the COVID-19 outbreak were negative, demonstrating a 100% specificity. The area under the curve, as evaluated by Receiver Operating Characteristic (ROC) analysis reached 0.998 (95%CI: 0.993-1) for samples taken from 11 days after symptoms onset. The kinetics of antibody responses were monitored after a first and second vaccination using serially collected DBS from 14 individuals. AbPEA offers highly specific and sensitive solution-phase antibody detection without requirement for secondary antibodies, no elution step when using DBS sample in a simple procedure that lends itself to multiplex survey of antibody responses.

A luciferase based automated assay for rapid and sensitive detection of SARS-CoV-2 antibodies.

The Coronavirus disease 2019 (COVID-19) pandemic brings great challenges to the public health and social economics around the world. As the pandemic continues and the mass vaccination goes on, monitoring the antibodies is particularly important for the epidemiological survey and vaccine assessment. Here, we developed a luciferase immunoprecipitation assay combined with an automated platform to detect anti-Receptor Binding Domain (RBD) antibody, where protein A and protein G modified magnetic beads were used to capture antibodies in serum samples and SARS-CoV-2 RBD was fused with Gaussia luciferase to label the captured target antibodies. The whole detection procedure can be completed within 20 min. The developed assay has proven up to 32 times more sensitive than ELISA for the detection of RBD antibodies. Furthermore, the results of the antibody detection of sera from vaccination as well as convalescence displayed good performance. The automated platform may provide a powerful tool for the control of COVID-19 pandemic by vaccination and the research of SARS-CoV-2 seroconversion.

Ultrarapid and ultrasensitive detection of SARS-CoV-2 antibodies in COVID-19 patients via a 3D-printed nanomaterial-based biosensing platform.

Rapid detection of antibodies during infection and after vaccination is critical for the control of infectious outbreaks, understanding immune response, and evaluating vaccine efficacy. In this manuscript, we evaluate a simple ultrarapid test for SARS-CoV-2 antibodies in COVID-19 patients, which gives quantitative results (i.e., antibody concentration) in 10-12 s using a previously reported nanomaterial-based three-dimensional (3D)-printed biosensing platform. This platform consists of a micropillar array electrode fabricated via 3D printing of aerosolized gold nanoparticles and coated with nanoflakes of graphene and specific SARS-CoV-2 antigens, including spike S1, S1 receptor-binding domain (RBD) and nucleocapsid (N). The sensor works on the principle of electrochemical transduction, where the change of sensor impedance is realized by the interactions between the viral proteins attached to the sensor electrode surface and the antibodies. The three sensors were used to test samples from 17 COVID-19 patients and 3 patients without COVID-19. Unlike other serological tests, the 3D sensors quantitatively detected antibodies at a concentration as low as picomole within 10-12 s in human plasma samples. We found that the studied COVID-19 patients had higher concentrations of antibodies to spike proteins (RBD and S1) than to the N protein. These results demonstrate the enormous potential of the rapid antibody test platform for understanding patients' immunity, disease epidemiology and vaccine efficacy, and facilitating the control and prevention of infectious epidemics.

Impaired Humoral Response to Second Dose of BNT162b2 Messenger RNA COVID-19 Vaccine in Kidney Transplant Recipients in Japan: Prospective Observational Study

Purpose: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has a major impact on solid organ transplant (SOT) recipients, with mortality rate up to 22%. The effect of SARS-CoV-2 vaccination are known to have poor responses even after 2nddose in SOT in Europe and the United States. We investigated immune response by detecting SARS-CoV-2 antibody (Ab) post two-doses of messenger RNA (mRNA)-based SARS-CoV-2 vaccinesin Japanese renal transplant recipients as prospective observational study. Method(s): 352 recipients who have no history of COVID-19, were confirmed no SARS-CoV-2 antibody before vaccination and received 2nd dose of BNT162b2 mRNA vaccine are enrolled in this study.Antibody detection test was performed by using the Roche Elecsys Anti-SARS-CoV-2 immunoassay after more than 4weeks following 2nddose of it. Negative for N-Ab (0.4U/ml>) and positive for S-Ab (0.8U/ml<) considered to be positive for SARS-CoV-2 Ab.As a healthy control, SARS-CoV-2 Ab was determined in 990 healthy volunteers (HV) as well as 98 kidney donors as control for chronic kidney disease (Donor). Result(s): The rate of positive for S-Ab was 56.2% in recipients while that was 100% in HV and Donor. Titer of S-Ab (U/ml) was 77 in recipients although that was 2400 in HV and 1100 in Donor, respectively, which indicating significantly lower in recipients. (Fig. 1) Interestingly, positive rate for s-Ab by detecting time following 2ndvaccination in recipients was 44% in 4-6 weeks, 54% in 6-8 weeks, 67% in 8-10 weeks, 72% in 10-12 weeks, 80% in 12-weeks, which suggesting that recipients have delayed response to 2ndvaccination while that was 100% in any time point in HV and Donor. (Fig.2) Moreover, to elucidate difference between responder (n=198) and non-responder (n=154) in recipients, we compared clinical background that influencedoutcome. In non-responder, there were significant difference in older age at vaccination, less lymphocyte, previousdoses of rituximab, concomitant use of mycophenolate mofetil and oral administration of more than three immunosuppressants. (Table.1) Conclusion(s): Kidney recipients have delayed response with lower titer of SARSCoV- 2 antibodyfollowing second dose of mRNA-based COVID-19 Vaccine.

Evaluation of two different domestic colloidal gold assay kit fornew coronavirus antibody in low endemic areas

This study aimed to evaluate the application and diagnostic efficacy of two different colloidal gold kits for the detection of 2019-nCoV immunoglobulin M antibody (anti-IgM) and immunoglobulin G antibody (anti-IgG) in Beijing, a low endemic area, and to guide the rational clinical application. The sera of 29 patients with confirmed novel coronavirus pneumonia (COVID-19) and 19 411 patients from the non-infected screening population were selected to evaluate the sensitivity, specificity and false-positive rate of the 2019-nCoV antibody test kits from Zhuhai Lizhu and Tangshan Innotek using colloidal gold immunochromatography. The sensitivity of Inotec 2019-nCoV was slightly higher than that of Lizhu 2019-nCoV, with a sensitivity of 58.62% and 55.17%, respectively;the specimen collection time of the all-negative group was significantly less than that of the antibody-positive group (P < 0.05);the false-positive rate of the two reagents in the low-prevalence area was 0.16%, and the false-positive rate of 2019-nCoV IgG was higher in Inotec than in Lizhu. The false positive rate for 2019-nCoV IgM was significantly higher than that for IgG for the same brand (Inotec ?2=14.756 09, P=0.000 0;Lizhu ?2=27.492 62, P=0.000). Conclusion The 2019-nCoV antibody test is rapid, simple and easy to perform, with high specificity, and can be used as a rapid screening indicator for new crowns;the specificity, correctness and negative predictive value of the two kits are good, and the application of the other kit for retesting when a positive result occurs can reduce the false positive rate of informing the clinic;the application and analysis of positive reports of new crown antibodies should be combined with the endemic area and clinical comprehensive judgment.

Using Split Luminescent Biosensors for SARS-CoV-2 Antibody Detection in Serum, Plasma, and Blood Samples.

Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID-19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme-linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS-CoV-2 virus to enable luminescent-based detection of spike- or nucleocapsid-binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low-/medium-throughput scale using plate-based assays, high-throughput scale using robotics, and point-of-care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid-handling robotics and in a point-of-care setting using a handheld, battery-powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point-of-care, nonlaboratory settings. © 2022 Wiley Periodicals LLC. Basic Protocol: SARS-CoV-2 antibody detection using the split-luciferase assay on a medium-throughput scale with a laboratory luminometer Alternate Protocol 1: High-throughput-based protocol for SARS-CoV-2 antibody detection using a robotic platform Alternate Protocol 2: Point-of-care-based protocol for SARS-CoV-2 antibody detection using a handheld luminometer Support Protocol: Determining positive/negative cutoffs for test samples and standardizing the assay between days.