Pig Sera-derived Anti-SARS-CoV-2 Antibodies with Controlled Specificity for Competitive Immunoassay

Ji-Hong BONG,Jaeyong JUNG,Jeong Soo SUNG,Chang Kyu LEE,Jae-Chul PYUN 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.4

Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies against nucleoprotein (NP) were purified from pig sera through two steps: (1) isolation of anti-SARS-CoV-2 NP antibodies using magnetic beads with immobilized SARS-CoV-2 NP and (2) filtration of anti-SARS-CoV-2 spike protein (SP). The binding activities of the isolated antibodies were confirmed using immunoassay and immunostaining. The binding constant (K_d) of the purified anti-SARS-CoV-2 NP antibodies was estimated using a surface plasmon resonance biosensor (SPR) and calculated to be 185 pM. From the dose-response curve, the limit of detection was estimated to be 1.02 pM. Finally, a competitive immunoassay was developed for detecting SARS-CoV-2 NP as well as SARS-CoV-2 in the culture fluid using magnetic beads with immobilized anti-SARS-CoV-2 NP antibodies. The results demonstrated (a) the detection of SARS-CoV-2 in viral fluid and (b) the discrimination of SARS-CoV-2 from SARS-CoV, MERS-CoV, and CoV strain 229E in viral fluids. Additionally, anti-SARS-CoV-2 antibodies against SP were obtained when the isolation process was performed using the magnetic beads with immobilized human SARS-CoV-2 SP.

Recombinant ACE2-Ig Fusion Protein Neutralizes SARS-CoV-2

Jin-Woo Song,고영상 대한미생물학회 2022 Journal of Bacteriology and Virology Vol.52 No.1

A novel coronavirus (SARS-CoV-2) is the causative agent for the COVID-19 pandemic, and needs for effective antiviral drugs have been demanded. Angiotensin-converting enzyme 2 (ACE2) has been shown to be a cellular receptor for SARS-CoV and SARS-CoV-2. For the therapeutic validation of ACE2, a recombinant protein consisting of the ACE2 extracellular domain fused to the IgG1 Fc domain (ACE2-Ig) was generated. ACE2-Ig exhibits proper pharmacological properties and binds with a high affinity to SARS-CoV and SARS-CoV-2 receptor-binding domain. Further, it neutralizes virus with SARS-CoV and SARS-CoV-2 spike proteins. Taken overall, these data suggest that ACE2-Ig has substantial anti-SARS-CoV-2 properties and warrant further study concerning its potential applications for the treatment of SARS-CoV-2.ㅍㅍㅍㅍㅍㅍ A novel coronavirus (SARS-CoV-2) is the causative agent for the COVID-19 pandemic, and needs for effective antiviral drugs have been demanded. Angiotensin-converting enzyme 2 (ACE2) has been shown to be a cellular receptor for SARS-CoV and SARS-CoV-2. For the therapeutic validation of ACE2, a recombinant protein consisting of the ACE2 extracellular domain fused to the IgG1 Fc domain (ACE2-Ig) was generated. ACE2-Ig exhibits proper pharmacological properties and binds with a high affinity to SARS-CoV and SARS-CoV-2 receptor-binding domain. Further, it neutralizes virus with SARS-CoV and SARS-CoV-2 spike proteins. Taken overall, these data suggest that ACE2-Ig has substantial anti-SARS-CoV-2 properties and warrant further study concerning its potential applications for the treatment of SARS-CoV-2.

Body Weight is Inversely Associated with Anti-SARS-CoV-2 Antibody Levels after BNT162b2 mRNA Vaccination in Young and Middle Aged Adults

Nam Su Youn,Jeon Seong Woo,Jung Deuk Kju,Heo Sung-Jae 대한감염학회 2022 Infection and Chemotherapy Vol.54 No.3

Background This study aimed to determine factors affecting serum levels of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies 2 months after coronavirus disease 2019 (COVID-19) vaccination in young and middle aged healthy adults. Materials and Methods Healthcare workers who have no history of SARS-CoV-2 infection, were enrolled at 2 months after second shot of BNT162b2 mRNA COVID-19 vaccine. Antibody immunoglobulin G against the spike protein subunit of SARS-CoV-2 was semi-quantitatively measured using 4 commercial enzyme-linked immunosorbent assay kits. Factors affecting anti-SARS-CoV-2 antibodies levels were investigated. Results Fifty-one persons (22 - 54 years, male sex; 19.6%) were enrolled and all participants acquired anti-SARS-CoV-2 antibodies in four diagnostic kits. Anti-SARS-CoV-2 antibodies were strongly correlated between diagnostic kits; SG Medical and Genscript (r = 0.942), SG Medical and HB Healthcare (r = 0.903), and HB Healthcare and Genscript (r = 0.868). We investigated factors affecting antibody level using SG medical kit. The median inhibition was 93.1%, and 84.0% of participants showed >90.0% inhibition. Systemic adverse event severity had no association with the anti-SARS-CoV-2 antibodies level. Antibody level was inversely correlated with weight (-0.312, P = 0.027), body mass index (BMI) (r = -0.303, P = 0.032), and body surface area (r = -0.285, P = 0.044). In multivariate analysis, the upper 50% of anti-SARS-CoV-2 antibodies (≥93.1%) was inversely associated with weight (odds ratio [OR]: 0.19; 95% confidence interval [CI]: 0.04 - 0.83 in weight ≥55kg) and BMI (OR: 0.12; 95% CI: 0.03 - 0.61 in BMI ≥22 kg/m2). Conclusion Anti-SARS-CoV-2 antibody was inversely correlated with weight and BMI, which may be used as a marker to predict immune response of BNT162b2 mRNA vaccination in young and middle aged adults. Trial Registration ClinicalTrials.gov Identifier: NCT05083026 Background This study aimed to determine factors affecting serum levels of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies 2 months after coronavirus disease 2019 (COVID-19) vaccination in young and middle aged healthy adults. Materials and Methods Healthcare workers who have no history of SARS-CoV-2 infection, were enrolled at 2 months after second shot of BNT162b2 mRNA COVID-19 vaccine. Antibody immunoglobulin G against the spike protein subunit of SARS-CoV-2 was semi-quantitatively measured using 4 commercial enzyme-linked immunosorbent assay kits. Factors affecting anti-SARS-CoV-2 antibodies levels were investigated. Results Fifty-one persons (22 - 54 years, male sex; 19.6%) were enrolled and all participants acquired anti-SARS-CoV-2 antibodies in four diagnostic kits. Anti-SARS-CoV-2 antibodies were strongly correlated between diagnostic kits; SG Medical and Genscript (r = 0.942), SG Medical and HB Healthcare (r = 0.903), and HB Healthcare and Genscript (r = 0.868). We investigated factors affecting antibody level using SG medical kit. The median inhibition was 93.1%, and 84.0% of participants showed >90.0% inhibition. Systemic adverse event severity had no association with the anti-SARS-CoV-2 antibodies level. Antibody level was inversely correlated with weight (-0.312, P = 0.027), body mass index (BMI) (r = -0.303, P = 0.032), and body surface area (r = -0.285, P = 0.044). In multivariate analysis, the upper 50% of anti-SARS-CoV-2 antibodies (≥93.1%) was inversely associated with weight (odds ratio [OR]: 0.19; 95% confidence interval [CI]: 0.04 - 0.83 in weight ≥55kg) and BMI (OR: 0.12; 95% CI: 0.03 - 0.61 in BMI ≥22 kg/m2). Conclusion Anti-SARS-CoV-2 antibody was inversely correlated with weight and BMI, which may be used as a marker to predict immune response of BNT162b2 mRNA vaccination in young and middle aged adults. Trial Registration ClinicalTrials.gov Identifier: NCT05083026

Microsecond molecular dynamics simulations revealed the inhibitory potency of amiloride analogs against SARS-CoV-2 E viroporin

Jaber, Abdullah All,Chowdhury, Zeshan Mahmud,Bhattacharjee, Arittra,Mourin, Muntahi,Keya, Chaman Ara,Bhuyan, Zaied Ahmed Korea Genome Organization 2021 Genomics & informatics Vol.19 No.4

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes small envelope protein (E) that plays a major role in viral assembly, release, pathogenesis, and host inflammation. Previous studies demonstrated that pyrazine ring containing amiloride analogs inhibit this protein in different types of coronavirus including SARS-CoV-1 small envelope protein E (SARS-CoV-1 E). SARS-CoV-1 E has 93.42% sequence identity with SARS-CoV-2 E and shared a conserved domain NS3/small envelope protein (NS3_envE). Amiloride analog hexamethylene amiloride (HMA) can inhibit SARS-CoV-1 E. Therefore, we performed molecular docking and dynamics simulations to explore whether amiloride analogs are effective in inhibiting SARS-CoV-2 E. To do so, SARS-CoV-1 E and SARS-CoV-2 E proteins were taken as receptors while HMA and 3-amino-5-(azepan-1-yl)-N-(diaminomethylidene)-6-pyrimidin-5-ylpyrazine-2-carboxamide (3A5NP2C) were selected as ligands. Molecular docking simulation showed higher binding affinity scores of HMA and 3A5NP2C for SARS-CoV-2 E than SARS-CoV-1 E. Moreover, HMA and 3A5NP2C engaged more amino acids in SARS-CoV-2 E. Molecular dynamics simulation for 1 ㎲ (1,000 ns) revealed that these ligands could alter the native structure of the proteins and their flexibility. Our study suggests that suitable amiloride analogs might yield a prospective drug against coronavirus disease 2019.

Diagnostic Techniques for SARS-CoV-2 Detection

Jong-Sik Kim(김종식),Na-Kyung Kang(강나경),Seon-Mi Park(박선미),Eun-Joo Lee(이은주),Kyung Tae Chung(정경태) 한국생명과학회 2020 생명과학회지 Vol.30 No.8

코로나바이러스감염증-19(COVID-19)는 SARS-CoV-2에 의해 발병된다. 지금까지 인간에게 감염되는 7 가지 종류의 코로나 바이러스가 보고되었다. 그 중, HCoV-229E, HCoV-OC43, HCoV-NL63, 그리고 HCoV-HKU1 등 4종류의 코로나바이러스는 감기와 같은 단순 호흡기 질환을 유발한다고 보고되었다. 반면, SARS-CoV는 2002년에, MERS-CoV는 2012년에 각각 대유행을 일으킨 바 있다. 가장 최근에는 2019년 12월 중국 우한에서 처음 보고된 SARS-CoV-2가 전세계적인 대유행의 원인이 되고 있다. 이러한 SARS-CoV-2를 진단하고, 치료하고, 예방하기 위해서는 신속 정확한 진단키트, 치료제, 그리고 안전한 백신의 개발의 필수적으로 요구된다. 이러한 강력한 도구들을 개발하기 위해서는 SARS-CoV-2의 표현형, 유전자형, 그리고 생활주기 등의 연구가 선행되어야 한다. SARSCoV-2의 진단기술은 현재 크게 두가지의 큰 분야인 분자진단과 면역혈청학적 진단으로 구분할 수 있다. 분자진단의 경우 SARS-CoV-2의 유전체를 대상으로 하며, 면역혈청학적 진단은 SARS-CoV-2의 항원 단백질 혹은 SARSCoV-2에 대한 항체를 대상으로 한다. 본 총설에서는 SARS-CoV-2의 표현형, 유전체 구조, 그리고 유전자 발현에 대해서 정리하고, SARS-CoV-2에 대한 다양한 진단 기술 등에 대한 기초지식을 제공하고자 한다. Coronavirus disease 19 (COVID-19) is caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome- Coronavirus 2). To date, seven coronaviruses that can infect humans were reported. Among them, infections with four coronavirus strains (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) resulted in mild symptoms such as common cold, whereas SARS-CoV and MERS-CoV caused severe symptoms and epidemics in 2002 and 2012, respectively. In the most recent, SARS-CoV-2 was first reported in Wuhan, China in December 2019 and became a notorious cause of the ongoing global pandemics. To diagnose, treat, and prevent COVID-19, the development of rapid and accurate diagnostic tools, specific therapeutic drugs, and safe vaccines essentially are required. In order to develop these powerful tools, it is prerequisite to understand a phenotype, a genotype, and life cycle of SARS-CoV-2. Diagnostic techniques have been developing rapidly around world and many countries take the fast track system to accelerate approval. Approved diagnostic devices are rapidly growing facing to urgent demand to identify carriers. Currently developed commercial diagnostic devices are divided into mainly two categories: molecular assay and serological & immunological assay. Molecular assays begins the reverse transcription step following polymerase chain reaction or isothermal amplification. Immunological assay targets SARS-CoV-2 antigen or anti-SARS-CoV-2 antibody of samples. In this review, we summarize the phenotype, genome structure and gene expression of SARS-CoV-2 and provide the knowledge on various diagnostic techniques for SARS-CoV-2.

Performance comparison between Elecsys Anti-SARS-CoV-2 and Anti-SARS-CoV-2 S and Atellica IM SARS-CoV-2 Total and SARS-CoV-2 IgG assays

Jeong Seri,홍유라,Hwang Hyunyong 고신대학교(의대) 고신대학교 의과대학 학술지 2022 고신대학교 의과대학 학술지 Vol.37 No.2

Background: Although serological severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests from several manufacturers have been introduced in South Korea and some are commercially available, the performance of these test kits has not yet been sufficiently validated. Therefore, we compared the performance of Elecsys Anti-SARS-CoV-2 (ACOV2) and Anti-SARS-CoV-2 S (ACOV2S) and Atellica IM SARS-CoV-2 Total (COV2T) and SARS-CoV-2 IgG (sCOVG) serological tests in this study.Methods: A total of 186 patient samples were used. For each test, we analyzed the positive rate of serological antibody tests, precision, linearity, and agreement among the four assays.Results: The positive rates of COV2T, sCOVG, and ACOV2S were high (81.7%–89.2%) in total, with those for ACOV2S being the highest, while those of ACOV2 were as low as 44.6%. This may be related to the high completion rate of vaccination in Korea. The repeatability and within-laboratory coefficients of variation were within the claimed allowable imprecision; however, further research is needed to establish an allowable imprecision at low concentrations. COV2T showed a linear fit, whereas sCOVG and ACOV2S were appropriately modeled with a nonlinear fit. Good agreement was found among COV2T, sCOVG, and ACOV2S; however, the agreement between ACOV2 and any one of the other methods was poor.Conclusions: Considering the different antigens used in serological SARS-CoV-2 antibody assays, the performance of the tested assays is thought to show no significant difference for the qualitative detection of antibodies to SARS-CoV-2.

Feature selection for effective prediction of SARS-COV-2 using machine learning

Punacha Gagan,Adiga Rama 한국유전학회 2024 Genes & Genomics Vol.46 No.3

Background With rise in variants of SARS-CoV-2, it is necessary to classify the emerging SARS-CoV-2 for early detection and thereby reduce human transmission. Genomic and proteomic information have less frequently been used for classifying in a machine learning (ML) approach for detection of SARS-CoV-2. Objective With this aim we used nucleoprotein and viral proteomic evolutionary information of SARS-CoV-2 along with the charge and basicity distribution of amino acids from various strains of SARS-CoV-2 to generate a disease severity model based on ML. Methods All sequence and clinical data were obtained from GISAID. Proteomic level calculations were added to comprise the dataset. The training set was used for feature selection. Select K- Best feature selection method was employed which was cross validated with testing set and performance evaluated. Delong’s test was also done. We also employed BIRCH clustering on SARS-CoV-2 for clustering the strains. Results Out of six ML models four were successful in training and testing. Extra Trees algorithm generated a micro-averaged F1-score of 74.2% and a weighted averaged area under the receiver operating characteristic curve (AUC-ROC) score of 73.7% with multi-class option. The feature selection set to 5, enhanced the ROC AUC from 73.7 to 76.4%. Accuracy of the selected model of 86.9% was achieved. Conclusion The unique features identified in the ML approach was able to classify disease severity into classes and had potential for predicting risk in newer variants. Background With rise in variants of SARS-CoV-2, it is necessary to classify the emerging SARS-CoV-2 for early detection and thereby reduce human transmission. Genomic and proteomic information have less frequently been used for classifying in a machine learning (ML) approach for detection of SARS-CoV-2. Objective With this aim we used nucleoprotein and viral proteomic evolutionary information of SARS-CoV-2 along with the charge and basicity distribution of amino acids from various strains of SARS-CoV-2 to generate a disease severity model based on ML. Methods All sequence and clinical data were obtained from GISAID. Proteomic level calculations were added to comprise the dataset. The training set was used for feature selection. Select K- Best feature selection method was employed which was cross validated with testing set and performance evaluated. Delong’s test was also done. We also employed BIRCH clustering on SARS-CoV-2 for clustering the strains. Results Out of six ML models four were successful in training and testing. Extra Trees algorithm generated a micro-averaged F1-score of 74.2% and a weighted averaged area under the receiver operating characteristic curve (AUC-ROC) score of 73.7% with multi-class option. The feature selection set to 5, enhanced the ROC AUC from 73.7 to 76.4%. Accuracy of the selected model of 86.9% was achieved. Conclusion The unique features identified in the ML approach was able to classify disease severity into classes and had potential for predicting risk in newer variants.

Comparative genetic analyses of Korean bat coronaviruses with SARS-CoV and the newly emerged SARS-CoV-2

Eun-Jee Na,Sook-Young Lee,김학준,Jae-Ku Oem 대한수의학회 2021 Journal of Veterinary Science Vol.22 No.1

Background: Bats have been considered natural reservoirs for several pathogenic human coronaviruses (CoVs) in the last two decades. Recently, a bat CoV was detected in the Republic of Korea; its entire genome was sequenced and reported to be genetically similar to that of the severe acute respiratory syndrome CoV (SARS-CoV). Objectives: The objective of this study was to compare the genetic sequences of SARS-CoV, SARS-CoV-2, and the two Korean bat CoV strains 16BO133 and B15-21, to estimate the likelihood of an interaction between the Korean bat CoVs and the human angiotensin-converting enzyme 2 (ACE2) receptor. Methods: The phylogenetic analysis was conducted with the maximum-likelihood (ML) method using MEGA 7 software. The Korean bat CoVs receptor binding domain (RBD) of the spike protein was analyzed by comparative homology modeling using the SWISS-MODEL server. The binding energies of the complexes were calculated using PRODIGY and MM/GBGA. Results: Phylogenetic analyses of the entire RNA-dependent RNA polymerase, spike regions, and the complete genome revealed that the Korean CoVs, along with SARS-CoV and SARS-CoV-2, belong to the subgenus Sarbecovirus, within BetaCoVs. However, the two Korean CoVs were distinct from SARS-CoV-2. Specifically, the spike gene of the Korean CoVs, which is involved in host infection, differed from that of SARS-CoV-2, showing only 66.8%–67.0% nucleotide homology and presented deletions within the RBD, particularly within regions critical for cross-species transmission and that mediate interaction with ACE2. Binding free energy calculation revealed that the binding affinity of Korean bat CoV RBD to hACE2 was drastically lower than that of SARS-CoV and SARS-CoV-2. Conclusions: These results suggest that Korean bat CoVs are unlikely to bind to the human ACE2 receptor.

Relative synonymous codon usage of ORF1ab in SARS-CoV-2 and SARS-CoV

Li Gun,Zhang Liang,Du Ning 한국유전학회 2021 Genes & Genomics Vol.43 No.11

Background COVID-19, as a novel coronavirus disease caused by new coronavirus SARS-CoV-2, spreads all over the world, and brings harm to human in many countries. Humans sufered a lot from both SARS-CoV-2 now and by SARS-CoV in the year 2003. It is important to understand the diferences and the relationships between these two types of viruses. Objective To compare relative synonymous codon usage of ORF1ab gene in SARS-CoV-2 and SARS-CoV, relative synonymous codon usage of their genomes are studied in this paper from the bioinformatics perspective. Methods The ORF1ab gene, which is an important non-structural polyprotein coding gene and now used for nucleic acid detection markers in many measurement method, in both SARS-CoV-2 (30 strains) and SARS-CoV (20 strains) are considered to be the research object in the present paper. The relative synonymous codon usage values of the ORF1ab gene are calculated to characterize the diferences and the evolutionary characteristics among 50 strains. Results There is a signifcant diference between SARS-CoV and SARS-CoV-2 when the relative synonymous codon usage value of ORF1ab genes is concerned. The results suggest that codon usage pattern of SARS-CoV is more similar to human than that of the SARS-CoV-2, and that the inner diference in SARS-CoV-2 strains is larger than that of SARS-CoV, which denote the larger diversity exits in the SARS-CoV-2 virus. Conclusion These results show that the relative synonymous codon usage values in the coronavirus could be used for further research on their evolutionary phenomenon.

Competitive Immunoassay of SARS‑CoV‑2 Using Pig Sera‑Derived Anti‑SARS‑CoV‑2 Antibodies

봉지홍,김태훈,정재용,이수정,성정수,이창규,강민정,김현옥,변재철 한국바이오칩학회 2021 BioChip Journal Vol.15 No.1

Anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) nucleoprotein (NP) antibodies were isolated from pig sera using human SARS-CoV-2 NP-immobilized magnetic beads. The binding properties of the isolated antibodies against SARS-CoV-2 NP were tested via flow cytometry using SARS-CoV-2 NP-immobilized magnetic beads. A competitive immunoassay was developed for detecting SARS-CoV-2 NP as well as SARS-CoV-2 in the culture fluid using magnetic beads with immobilized anti-SARS-CoV-2 NP antibodies. Selectivity tests were carried out during the competitive immunoassay for SARS-CoV, MERS-CoV, and CoV strain 229E in the culture fluid.