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1.
Comput Struct Biotechnol J ; 20: 4501-4516, 2022.
Article in English | MEDLINE | ID: covidwho-2076032

ABSTRACT

Emerging SARS-CoV-2 variants with higher transmissibility and immune escape remain a persistent threat across the globe. This is evident from the recent outbreaks of the Delta (B.1.617.2) and Omicron variants. These variants have originated from different continents and spread across the globe. In this study, we explored the genomic and structural basis of these variants for their lineage defining mutations of the spike protein through computational analysis, protein modeling, and molecular dynamic (MD) simulations. We further experimentally validated the importance of these deletion mutants for their immune escape using a pseudovirus-based neutralization assay, and an antibody (4A8) that binds directly to the spike protein's NTD. Delta variant with the deletion and mutations in the NTD revealed a better rigidity and reduced flexibility as compared to the wild-type spike protein (Wuhan isolate). Furthermore, computational studies of 4A8 monoclonal antibody (mAb) revealed a reduced binding of Delta variant compared to the wild-type strain. Similarly, the MD simulation data and virus neutralization assays revealed that the Omicron also exhibits immune escape, as antigenic beta-sheets appear to be disrupted. The results of the present study demonstrate the higher possibility of immune escape and thereby achieved better fitness advantages by the Delta and Omicron variants, which warrants further demonstrations through experimental evidences. Our study, based on in-silico computational modelling, simulations, and pseudovirus-based neutralization assay, highlighted and identified the probable mechanism through which the Delta and Omicron variants are more pathogenically evolved with higher transmissibility as compared to the wild-type strain.

2.
Carbohydrate Polymers ; 299:120173, 2023.
Article in English | ScienceDirect | ID: covidwho-2060487

ABSTRACT

COVID-19 caused by SARS-CoV-2 has spread around the world at an unprecedented rate. A more homogeneous oligo-porphyran with mean molecular weight of 2.1 kD, named OP145, was separated from Pyropia yezoensis. NMR analysis showed OP145 was mainly composed of →3)-β-d-Gal-(1 → 4)-α-l-Gal (6S) repeating units with few replacement of 3,6-anhydride, and the molar ratio was 1:0.85:0.11. MALDI-TOF MS revealed OP145 contained mainly tetrasulfate-oligogalactan with Dp range from 4 to 10 and with no more than two 3,6-anhydro-α-l-Gal replacement. The inhibitory activity of OP145 against SARS-CoV-2 was investigated in vitro and in silico. OP145 could bind to Spike glycoprotein (S-protein) through SPR result, and pseudovirus tests confirmed that OP145 could inhibite the infection with an EC50 of 37.52 μg/mL. Molecular docking simulated the interaction between the main component of OP145 and S-protein. All the results indicated that OP145 had the potency to treat and prevent COVID-19.

3.
Viruses ; 14(9)2022 09 16.
Article in English | MEDLINE | ID: covidwho-2043976

ABSTRACT

Neutralization assays are experimental surrogates for the effectiveness of infection- or vaccine-elicited polyclonal antibodies and therapeutic monoclonal antibodies targeting SARS-CoV-2. However, the measured neutralization can depend on the details of the experimental assay. Here, we systematically assess how ACE2 expression in target cells affects neutralization by antibodies to different spike epitopes in lentivirus pseudovirus neutralization assays. For high ACE2-expressing target cells, receptor-binding domain (RBD) antibodies account for nearly all neutralizing activity in polyclonal human sera. However, for lower ACE2-expressing target cells, antibodies targeting regions outside the RBD make a larger (although still modest) contribution to serum neutralization. These serum-level results are mirrored for monoclonal antibodies: N-terminal domain (NTD) antibodies and RBD antibodies that do not compete for ACE2 binding incompletely neutralize on high ACE2-expressing target cells, but completely neutralize on cells with lower ACE2 expression. Our results show that the ACE2 expression level in the target cells is an important experimental variable, and that high ACE2 expression emphasizes the role of a subset of RBD-directed antibodies.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Epitopes , Humans , Spike Glycoprotein, Coronavirus
4.
Swiss Medical Weekly ; 152:10S, 2022.
Article in English | EMBASE | ID: covidwho-2040808

ABSTRACT

Progress in the fight against COVID-19 is jeopardized by the emergence of SARS-CoV-2 variants that diminish or abolish the efficacy of vaccines and antiviral monoclonal antibodies. Novel immune therapies are therefore needed, that are broadly effective against present and future coronavirus threats. In principle, this could be achieved by focusing on portions of the virus that are both functionally relevant and averse to change. The Subdomain 1 (SD1) of SARS-CoV-2 Spike protein is adjacent to the RBD and its sequence is conserved across SARS-CoV-2 variants, except for substitutions A570D in Alpha (B.1.1.7) and T547K in Omicron BA.1 (B.1.1.529). In order to specifically identify and study human antibodies targeting SD1, we designed a flow cytometry-based strategy that combines negative selection of B cells binding to the Receptor Binding Domain (RBD) with positive selection of those binding to SD1-RBD fusion protein. Among the 15 produced human monoclonal antibodies, 6 are SD1-specific. 3 of them cross-react with SD1-RBDs corresponding to all six variants of concern and 2 are neutralizing SARS-CoV-2 pseudovirus. Antibody sd1.040 also neutralizes Delta, Omicron BA.1 and Omicron BA.2 pseudovirus, synergizes with an antibody to the RBD for neutralization, and protects mice when present in a bispecific antibody. Thus, naturally occurring antibodies can neutralize SARS-CoV-2 variants by binding to SD1 and can act synergistically against SARS-CoV-2 in preclinical models.

5.
J Biol Chem ; 298(11): 102500, 2022 Sep 22.
Article in English | MEDLINE | ID: covidwho-2041895

ABSTRACT

Coronavirus disease represents a real threat to the global population, and understanding the biological features of the causative virus, that is, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is imperative for mitigating this threat. Analyses of proteins such as primary receptors and coreceptors (cofactors), which are involved in the entry of SARS-CoV-2 into host cells, will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates present in proximity to the attachment sites of SARS-CoV-2 spike proteins, using proximity labeling and proteomic analysis. The identified proteins represent key candidate factors that may be required for viral entry. We found SARS-CoV-2 host protein DPP4, cell adhesion protein Cadherin 17, and glycoprotein CD133 colocalized with cell membrane-bound SARS-CoV-2 spike proteins in Caco-2 cells and thus showed potential as candidate factors. Additionally, our analysis of the experimental infection of HEK293T cells with a SARS-CoV-2 pseudovirus indicated a 2-fold enhanced infectivity in the CD133-ACE2-coexpressing HEK293T cells compared to that in HEK293T cells expressing ACE-2 alone. The information and resources regarding these coreceptor labeling and analysis techniques could be utilized for the development of antiviral agents against SARS-CoV-2 and other emerging viruses.

6.
Biosaf Health ; 4(5): 321-329, 2022 Oct.
Article in English | MEDLINE | ID: covidwho-2007566

ABSTRACT

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to unprecedented social and economic disruption. Many nucleic acid testing (NAT) laboratories in China have been established to control the epidemic better. This proficiency testing (PT) aims to evaluate the participants' performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities. Two different concentrations of RNA samples (A, B) were used for quantitative PT. Pseudovirus samples D, E (different concentrations) and negative sample (F) were used for qualitative PT. 50 data sets were reported for qualitative PT, of which 74.00% were entirely correct for all samples. Forty-two laboratories participated in the quantitative PT. 37 submitted all gene results, of which only 56.76% were satisfactory. For qualitative detection, it is suggested that laboratories should strengthen personnel training, select qualified detection kits, and reduce cross-contamination to improve detection accuracy. For quantitative detection, the results of the reverse transcription digital PCR (RT-dPCR) method were more comparable and reliable than those of reverse transcription quantitative PCR (RT-qPCR). The copy number concentration of ORF1ab and N in samples A and B scattered in 85, 223, 50, and 106 folds, respectively. The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills. Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing, 95.65% of the laboratories with satisfactory quantitative results also judged the qualitative results correctly, while 85.71% of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments. Therefore, the quantitative ability is the basis of qualitative judgment. Overall, participants from hospitals reported more satisfactory results than those from enterprises and universities. Therefore, surveillance, daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.

7.
Vaccine X ; 12: 100211, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-2004302

ABSTRACT

Background: Heterologous prime-boost SARS-CoV-2 vaccination is a widely accepted strategy during the COVID-19 pandemic, which generated a superior immune response than homologous vaccination strategy. Objective: To describe immunogenicity of heterologous prime-boost vaccination with inactivated vaccine, CoronaVac, followed by BNT162b2 and 5-month booster dose with BNT162b2 in healthy Thai adolescents. Methods: Adolescents aged 12-18 years were randomized 1:1:1:1 to receive CoronaVac (SV) followed by BNT162b2 (PZ) 30 or 20 µg at either 3- or 6-week interval (SV3w/PZ30µg, SV3w/PZ20µg, SV6w/PZ30µg or SV6w/PZ20µg). During the Omicron-predominant period, participants were offered a BNT162b2 booster dose 30, 15, or 10 µg. Immunogenicity was determined using IgG antibody against spike-receptor-binding domain of wild type(anti-S-RBD IgG) and surrogate virus neutralization test(sVNT) against Delta variant at 14 days and 5 months after the 2nd dose. Neutralization tests(sVNT and pseudovirus neutralization test; pVNT) against Omicron strain were tested pre- and 14 days post-booster dose. Results: In October 2021, 76 adolescents with a median age of 14.3 years (IQR 12.7-16.0) were enrolled: 20 in SV3w/PZ30µg; 17 in SV3w/PZ20µg; 20 in SV6w/PZ30µg; 19 in SV6w/PZ20µg. At day 14, the geometric mean(GM) of anti-S-RBD IgG in SV3w/PZ30µg was 4713 (95 %CI 4127-5382) binding-antibody unit (BAU)/ml, while geometric mean ratio(GMR) was 1.28 (1.09-1.51) in SV6w/PZ30µg. The GMs of sVNT against Delta variants at day 14 among participants in SV3w/PZ30µg and SV6wk/PZ30µg arm were 95.3 % and 99.7 %inhibition, respectively. At 5 months, GMs of sVNT against Delta variants in SV3w/PZ30µg were significantly declined to 47.8 % but remained at 89.0 % inhibition among SV6w/PZ30µg arm. In April 2022, 52 adolescents received a BNT162b2 booster dose. Proportion of participants with sVNT against Omicron strain > 80 %inhibition was significantly increased from 3.8 % pre-booster to 67 % post-booster. Proportion of participants with pVNT ID50 > 185 was 42 % at 14 days post 2nd dose and 88 % post booster, respectively. Conclusions: Heterologous prime-boost vaccination with CoronaVac followed by BNT162b2 induced high neutralizing titer against SARS-CoV-2 Delta strain. After 5-month interval, booster with BNT162b2 induced high neutralizing titer against Omicron strain.Thai Clinical Trials Registry (thaiclinicaltrials.org): TCTR20210923012.

8.
Viruses ; 14(8)2022 08 16.
Article in English | MEDLINE | ID: covidwho-1988000

ABSTRACT

Coronaviruses isolated from bats and pangolins are closely related to SARS-CoV-2, the causative agent of COVID-19. These so-called sarbecoviruses are thought to pose an acute pandemic threat. As SARS-CoV-2 infection and vaccination have become more widespread, it is not known whether neutralizing antibodies to SARS-CoV-2 can cross-neutralize coronaviruses transmitted by bats or pangolins. In this study, we analyzed antibody-mediated neutralization with serum samples from COVID-19 patients (n = 31) and those immunized with inactivated SARS-CoV-2 vaccines (n = 20) against lentivirus-based pseudo-viruses carrying the spike derived from ancestral SARS-CoV-2, bat (RaTG13 or RshSTT182), or pangolin coronaviruses (PCoV-GD). While SARS-CoV-2, PCoV-GD, and RshSTT182 spikes could promote cell-cell fusion in VeroE6 cells, the RaTG13 spike did not. RaTG13, on the other hand, was able to induce cell-cell fusion in cells overexpressing ACE2. Dramatic differences in neutralization activity were observed, with the highest level observed for RaTG13, which was even significantly higher than SARS-CoV-2, PCoV-GD, and RshSTT182 pseudo-viruses. Interestingly, pseudo-viruses containing the chimeric protein in which the receptor-binding domain (RBD) of PCoV-GD spike was replaced by that of RaTG13 could be strongly neutralized, whereas those carrying RaTG13 with the RBD of PCoV-GD were significantly less neutralized. Because the high neutralizing activity against RaTG13 appears to correlate with its low affinity for binding to the human ACE2 receptor, our data presented here might shed light on how pre-existing immunity to SARS-CoV-2 might contribute to protection against related sarbecoviruses with potential spillover to the human host.


Subject(s)
COVID-19 , Chiroptera , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Pangolins , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
9.
Structure ; 30(10): 1432-1442.e4, 2022 10 06.
Article in English | MEDLINE | ID: covidwho-1967156

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and human coronavirus (hCoV)-NL63 utilize ACE2 as the functional receptor for cell entry, which leads to zoonotic infection. Horses (Equus caballus) attracted our attention because the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 and SARS-CoV-2-related coronaviruses bind equine ACE2 (eACE2) with high affinity. Here we show that eACE2 binds the RBDs of these three coronaviruses and also SARS-CoV-2 variants but with lower affinities compared with human ACE2 (hACE2). Structural analysis and mutation assays indicated that eACE2-H41 accounts for the lower binding affinity of eACE2 to the RBDs of SARS-CoV-2 variants (Alpha, Beta, and Gamma), SARS-CoV, and hCoV-NL63. Pseudovirus infection assays showed that the SARS-CoV-2 Delta strain (B.1.617.2) displayed a significantly increased infection efficiency in eACE2-expressing HeLa cells. Our results reveal the molecular basis of eACE2 binding to the RBDs of SARS-CoV, SARS-CoV-2, and hCoV-NL63, which provides insights into the potential animal transmission of these ACE2-dependent coronaviruses.


Subject(s)
COVID-19 , Coronavirus NL63, Human , Angiotensin-Converting Enzyme 2 , Animals , HeLa Cells , Horses , Humans , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
10.
Viruses ; 14(6)2022 06 18.
Article in English | MEDLINE | ID: covidwho-1964108

ABSTRACT

The global spread of SARS-CoV-2 and its variants poses a serious threat to human health worldwide. Recently, the emergence of Omicron has presented a new challenge to the prevention and control of the COVID-19 pandemic. A convenient and reliable in vitro neutralization assay is an important method for validating the efficiency of antibodies, vaccines, and other potential drugs. Here, we established an effective assay based on a pseudovirus carrying a full-length spike (S) protein of SARS-CoV-2 variants in the HIV-1 backbone, with a luciferase reporter gene inserted into the non-replicate pseudovirus genome. The key parameters for packaging the pseudovirus were optimized, including the ratio of the S protein expression plasmids to the HIV backbone plasmids and the collection time for the Alpha, Beta, Gamma, Kappa, and Omicron pseudovirus particles. The pseudovirus neutralization assay was validated using several approved or developed monoclonal antibodies, underscoring that Omicron can escape some neutralizing antibodies, such as REGN10987 and REGN10933, while S309 and ADG-2 still function with reduced neutralization capability. The neutralizing capacity of convalescent plasma from COVID-19 convalescent patients in Wuhan was tested against these pseudoviruses, revealing the immune evasion of Omicron. Our work established a practical pseudovirus-based neutralization assay for SARS-CoV-2 variants, which can be conducted safely under biosafety level-2 (BSL-2) conditions, and this assay will be a promising tool for studying and characterizing vaccines and therapeutic candidates against Omicron-included SARS-CoV-2 variants.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/therapy , Humans , Immunization, Passive , Neutralization Tests/methods , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
11.
Chin Med ; 17(1): 88, 2022 Jul 27.
Article in English | MEDLINE | ID: covidwho-1962860

ABSTRACT

BACKGROUND: Since the outbreak of COVID-19 has resulted in over 313,000,000 confirmed cases of infection and over 5,500,000 deaths, substantial research work has been conducted to discover agents/ vaccines against COVID-19. Undesired adverse effects were observed in clinical practice and common vaccines do not protect the nasal tissue. An increasing volume of direct evidence based on clinical studies of traditional Chinese medicines (TCM) in the treatment of COVID-19 has been reported. However, the safe anti-inflammatory and anti-fibrotic proprietary Chinese medicines nasal spray, designated as Allergic Rhinitis Nose Drops (ARND), and its potential of re-purposing for suppressing viral infection via SARS-CoV-2 RBD (Delta)- angiotensin converting enzyme 2 (ACE2) binding have not been elucidated. PURPOSE: To characterize ARND as a potential SARS-CoV-2 entry inhibitor for its possible preventive application in anti-virus hygienic agent. METHODS: Network pharmacology analysis of ARND was adopted to asacertain gene targets which were commonly affected by COVID-19. The inhibitory effect of ARND on viral infection was determined by an in vitro pseudovirus assay. Furthermore, ARND was confirmed to have a strong binding affinity with ACE2 and SARS-CoV-2 spike-RBD (Delta) by ELISA. Finally, inflammatory and fibrotic cell models were used in conjunction in this study. RESULTS: The results suggested ARND not only inhibited pseudovirus infection and undermined the binding affinity between ACE2 and the Spike protein (Delta), but also attenuated the inflammatory response upon infection and may lead to a better prognosis with a lower risk of pulmonary fibrosis. The data in this study also provide a basis for further development of ARND as an antiviral hygienic product and further investigations on ARND in the live virus, in vivo and COVID-19 patients. ARND holds promise for use in the current COVID-19 outbreak as well as in future pandemics. CONCLUSION: ARND could be considered as a safe anti-SARS-CoV-2 agent with potential to prevent SARS-CoV-2 coronavirus infection.

12.
J Ginseng Res ; 2022 Jul 15.
Article in English | MEDLINE | ID: covidwho-1936774

ABSTRACT

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean red ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

13.
iScience ; : 104739, 2022 Jul 09.
Article in English | MEDLINE | ID: covidwho-1926567

ABSTRACT

Several effective SARS-CoV-2 vaccines have been developed using different technologies. Although these vaccines target the isolates collected early in the pandemic, many have protected against serious illness from newer variants. Nevertheless, efficacy has diminished against successive variants and the need for effective and affordable vaccines persists especially in the developing world. Here, we adapted our protein-protein conjugate vaccine technology to generate a vaccine based on receptor binding domain (RBD) antigen. RBD was conjugated to a carrier protein, EcoCRM, to generate two types of conjugates: crosslinked and radial conjugates. In the crosslinked conjugate, antigen and carrier are chemically crosslinked; in the radial conjugate, antigen is conjugated to the carrier by site-specific conjugation. With AS01 adjuvant, both conjugates showed enhanced immunogenicity in mice compared to RBD, with a Th1 bias. In hACE2 binding inhibition and pseudovirus neutralization assays, sera from mice vaccinated with the radial conjugate demonstrated strong functional activity.

14.
Molecules ; 27(12)2022 Jun 13.
Article in English | MEDLINE | ID: covidwho-1911480

ABSTRACT

COVID-19, resulting from infection by the SARS-CoV-2 virus, caused a contagious pandemic. Even with the current vaccines, there is still an urgent need to develop effective pharmacological treatments against this deadly disease. Here, we show that the water and ethanol extracts of the root and rhizome of Polygonum cuspidatum (Polygoni Cuspidati Rhizoma et Radix), a common Chinese herbal medicine, blocked the entry of wild-type and the omicron variant of the SARS-CoV-2 pseudotyped virus into fibroblasts or zebrafish larvae, with IC50 values ranging from 0.015 to 0.04 mg/mL. The extracts were shown to inhibit various aspects of the pseudovirus entry, including the interaction between the spike protein (S-protein) and the angiotensin-converting enzyme II (ACE2) receptor, and the 3CL protease activity. Out of the chemical compounds tested in this report, gallic acid, a phytochemical in P. cuspidatum, was shown to have a significant anti-viral effect. Therefore, this might be responsible, at least in part, for the anti-viral efficacy of the herbal extract. Together, our data suggest that the extracts of P. cuspidatum inhibit the entry of wild-type and the omicron variant of SARS-CoV-2, and so they could be considered as potent treatments against COVID-19.


Subject(s)
COVID-19 , Fallopia japonica , Animals , Antiviral Agents/analysis , Antiviral Agents/pharmacology , COVID-19/drug therapy , Fallopia japonica/chemistry , Peptide Hydrolases , Plant Extracts/analysis , Plant Extracts/pharmacology , Rhizome/chemistry , SARS-CoV-2 , Viral Pseudotyping , Zebrafish
15.
Cell Struct Funct ; 47(1): 43-53, 2022 Jun 25.
Article in English | MEDLINE | ID: covidwho-1910415

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Key words: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.


Subject(s)
Spike Glycoprotein, Coronavirus , Vesicular stomatitis Indiana virus , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/biosynthesis , Vesicular stomatitis Indiana virus/metabolism
16.
Front Immunol ; 13: 901217, 2022.
Article in English | MEDLINE | ID: covidwho-1903025

ABSTRACT

Fc-mediated virus entry has been observed for many viruses, but the characterization of this activity in convalescent plasma against SARS-CoV-2 Variants of Concern (VOC) is undefined. In this study, we evaluated Fc-mediated viral entry (FVE) on FcγRIIa-expressing HEK293 cells in the presence of SARS-CoV-2 convalescent plasma and compared it with SARS-CoV-2 pseudovirus neutralization using ACE2-expressing HEK293 cells. The plasma were collected early in the pandemic from 39 individuals. We observed both neutralization and FVE against the infecting Washington SARS-CoV-2 strain for 31% of plasmas, neutralization, but not FVE for 61% of plasmas, and no neutralization or FVE for 8% of plasmas. Neutralization titer correlated significantly with the plasma dilution at which maximum FVE was observed, indicating Fc-mediated uptake peaked as neutralization potency waned. While total Spike-specific plasma IgG levels were similar between plasma that mediated FVE and those that did not, Spike-specific plasma IgM levels were significantly higher in plasma that did not mediate FVE. Plasma neutralization titers against the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) VOC were significantly lower than titers against the Washington strain, while plasma FVE activity against the VOC was either higher or similar. This is the first report to demonstrate a functional shift in convalescent plasma antibodies from neutralizing and FVE-mediating against the earlier Washington strain, to an activity mediating only FVE and no neutralization activity against the emerging VOC, specifically the Beta (B.1.351) and Gamma (P.1) VOC. It will be important to determine the in vivo relevance of these findings.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/therapy , HEK293 Cells , Humans , Immunization, Passive , Immunoglobulin Fc Fragments , Spike Glycoprotein, Coronavirus , Virus Internalization
17.
Phytomedicine ; 102: 154154, 2022 Jul 20.
Article in English | MEDLINE | ID: covidwho-1889764

ABSTRACT

BACKGROUND: Globally, COVID-19 has caused millions of deaths and led to unprecedented socioeconomic damage. There is therefore, in addition to vaccination, an urgent need to develop complementary effective treatments and/or protective and preventative therapies against this deadly disease. METHODS: Here, a multi-component testing platform was established to screen a library of herbal extracts from traditional Chinese medicine (TCM), to identify potent herbal extracts/phytochemicals as possible therapeutics for COVID-19. We utilized assays for spike protein (S-protein) binding to angiotensin-converting enzyme II (ACE2); the enzymatic inhibition of 3CL protease; and entry of the SARS-CoV-2 pseudovirus into cultured HEK293T cells and zebrafish larvae. RESULTS: Over a thousand herbal extracts were screened and approximately 20 positive hits were identified. Among these, we found that the water and ethanol extracts of Polygoni Multiflori Radix (PMR) significantly inhibited S-protein binding to ACE2, 3CL protease activity, and viral entry into the cell and fish models. The water extract was more effective than the ethanol extract, with IC50 values of 25 to 500 µg/ml. In addition, the polysaccharide-depleted fraction of the former, and epigallocatechin gallate (EGCG) which was found in both extracts, displayed significant antiviral activity. CONCLUSIONS: Our results indicate that the water and ethanol extracts of PMR have an inhibitory effect on SARS-CoV-2 pseudovirus host-cell entry. Furthermore, EGCG might be an active component of PMR, which blocks SARS-CoV-2 entry to cells. Taken together, our findings suggest that PMR might be considered as a potential treatment for COVID-19.


Subject(s)
COVID-19 , Polygonum , Angiotensin-Converting Enzyme 2 , Animals , COVID-19/drug therapy , Ethanol , HEK293 Cells , Humans , Larva , SARS-CoV-2 , Water , Zebrafish
18.
J Infect Dis ; 226(10): 1731-1742, 2022 Nov 11.
Article in English | MEDLINE | ID: covidwho-1886452

ABSTRACT

BACKGROUND: Messenger RNA (mRNA)-1273 vaccine demonstrated 93.2% efficacy against coronavirus disease 2019 (COVID-19) in the Coronavirus Efficacy (COVE) trial. The humoral immunogenicity results are now reported. METHODS: Participants received 2 mRNA-1273 (100 µg) or placebo injections, 28 days apart. Immune responses were evaluated in a prespecified, randomly selected per-protocol immunogenicity population (n = 272 placebo; n = 1185 mRNA-1273). Serum binding antibodies (bAbs) and neutralizing antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-spike protein were assessed at days 1, 29, and 57 by baseline SARS-CoV-2-negative (n = 1197) and SARS-CoV-2-positive (n = 260) status, age, and sex. RESULTS: SARS-CoV-2-negative vaccinees had bAb geometric mean AU/mL levels of 35 753 at day 29 that increased to 316 448 at day 57 and nAb inhibitory dilution 50% titers of 55 at day 29 that rose to 1081 at day 57. In SARS-CoV-2-positive vacinees, the first mRNA-1273 injection elicited bAb and nAb levels that were 11-fold (410 049) and 27-fold (1479) higher than in SARS-CoV-2-negative vaccinees, respectively, and were comparable to levels after 2 injections in uninfected participants. Findings were generally consistent by age and sex. CONCLUSIONS: mRNA-1273 elicited robust serologic immune responses across age, sex, and SARS-CoV-2 status, consistent with its high COVID-19 efficacy. Higher immune responses in those previously infected support a booster-type effect. Clinical Trials Registration. NCT04470427.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , 2019-nCoV Vaccine mRNA-1273 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Immunogenicity, Vaccine , RNA, Messenger , Spike Glycoprotein, Coronavirus
19.
China Tropical Medicine ; 22(3):240-245, 2022.
Article in Chinese | Scopus | ID: covidwho-1879662

ABSTRACT

Objective To construct SARS-CoV-2 pseudovirus, optimize its preparation protocol, and apply it to the evaluation of antibody neutralization activity. Methods The optimized sequence of spike (S) gene of SARS-CoV-2 was synthesized, the pseudovirus titers were measured, and the expressed S protein was then detected by Western blot. Finally, quantitative ELISA was used to measure the serum IgG antibody titers in recipients who had received either one or two doses of COVID-19 inactivated vaccine. Meanwhile, the sera were tested for their reactivity with the pseudovirus using neutralization tests. Results S gene was confirmed to have been successfully cloned into the vector, generating the pcDNA3.1-S plasmid. Co-transfection of pNL4-3.Luc.R-E- and pcDNA3.1-S at a ratio of 2∶1 could lead to higher packing efficacy and pseudovirus titers. Expression of the S protein was verified by Western blot. Moreover, this SARS-CoV-2 pseudovirus showed a broad host infectivity in Vero, Huh7.5, A549-hACE2 and 293T-hACE2 cells, with the highest relative luciferase unit (RLU) in 293T-hACE2. Comparing the IgG levels measured by ELISA in sera collected from COVID-19 vaccine recipients, we observed a higher titer in those who received two doses of inactivated vaccine (S/CO=10.27±3.33), measured one week after the second shot. However, the IgG level significantly dropped(S/CO=2.36±2.25)half year post-vaccination. Amongst the serum samples tested, one with an S/CO of 10.32 could successfully inhibit the infection of SARS-CoV-2 pseudovirus in 293T-hACE2 cells at a dilution of 1/1 066. Conclusion We have established a method for preparing the SARS-CoV-2 pseudovirus, which can be used for detection of the neutralizing antibodies and the evaluation of humoral immune response post-vaccination. © 2022 Editorial Office of Chinese Journal of Schistosomiasis Control. All Rights Reserved.

20.
Biomed Pharmacother ; 151: 113104, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1850705

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has continuously evolved, resulting in the emergence of several variants of concern (VOCs). To study mechanisms of viral entry and potentially identify specific inhibitors, we pseudotyped lentiviral vectors with different SARS-CoV-2 VOC spike variants (D614G, Alpha, Beta, Delta, Omicron/BA.1), responsible for receptor binding and membrane fusion. These SARS-CoV-2 lentiviral pseudoviruses were applied to screen 774 FDA-approved drugs. For the assay we decided to use CaCo2 cells, since they equally allow cell entry through both the direct membrane fusion pathway mediated by TMPRSS2 and the endocytosis pathway mediated by cathepsin-L. The active molecules which showed stronger differences in their potency to inhibit certain SARS-CoV-2 VOCs included antagonists of G-protein coupled receptors, like phenothiazine-derived antipsychotic compounds such as Chlorpromazine, with highest activity against the Omicron pseudovirus. In general, our data showed that the various VOCs differ in their preferences for cell entry, and we were able to identify synergistic combinations of inhibitors. Notably, Omicron singled out by relying primarily on the endocytosis pathway while Delta preferred cell entry via membrane fusion. In conclusion, our data provide new insights into different entry preferences of SARS-CoV-2 VOCs, which might help to identify new drug targets.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/drug therapy , Caco-2 Cells , Drug Evaluation, Preclinical , Humans , Spike Glycoprotein, Coronavirus/metabolism
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