Establishment of a pseudovirus neutralization assay based on SARS-CoV-2 S protein incorporated into lentiviral particles

AbstractThe aim of this study was to present and evaluate novel oral vaccines, based on self-amplifying RNA lipid nanparticles (saRNA LNPs), saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum, to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) variants alpha and delta. After invitro evaluation of the oral vaccines on HEK293T/17 cells, we found that saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum could express S-protein at both mRNA and protein levels. In the next step, BALB/c mice were orally vaccinated with saRNA LNPs, saRNA transfected Lactobacillus plantarum LNPs, and saRNA transfected Lactobacillus plantarum at weeks 1 and 3. Importantly, a high titer of IgG and IgA was observed by all of them, sharply in week 6 (P < 0.05). In all study groups, their ratio of IgG2a/IgG1 was upper 1, indicating Th1-biased responses. Wild-type viral neutralization assay showed that the secreted antibodies in vaccinated mice and recovered COVID-19 patients could neutralize SARS-COV-2 variants alpha and delta. After oral administration of oral vaccines, biodistribution assay was done. It was found that all of them had the same biodistribution pattern. The highest concentration of S-protein was seen in the small intestine, followed by the large intestine and liver.

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Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays

10.1101/2020.09.07.20188151 ◽

2020 ◽

Author(s):

Arantxa Valdivia ◽

Ignacio Torres ◽

Victor Latorre ◽

Carla Frances-Gomez ◽

Eliseo Albert ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Virus Neutralization ◽

Pseudotyped Virus ◽

S Protein ◽

Neutralizing Activity ◽

Virus Neutralization Assay ◽

Degree Correlation ◽

Antibody Titers

Background: Whether antibody levels measured by commercially-available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. Objectives: To evaluate the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 Spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Patients and Methods: Ninety sera from 51 hospitalized COVID-19 patients were assayed by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG and the COVID-19 ELISA IgG assays. Results: Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (Rho=0.73) and moderate for the remaining assays (Rho=0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. Conclusions: the suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.

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Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein

10.1101/2021.01.27.428534 ◽

2021 ◽

Author(s):

Jillian M. DiMuzio ◽

Baron C. Heimbach ◽

Raymond J. Howanski ◽

John P. Dowling ◽

Nirja B. Patel ◽

...

Keyword(s):

B Cell ◽

Protein A ◽

Humoral Response ◽

Viral Proteins ◽

Neutralization Assay ◽

Full Length ◽

Soluble Form ◽

Cell Repertoire ◽

Memory B Cell ◽

S Protein

AbstractPatients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.

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Middle East respiratory coronavirus (MERS-CoV) spike (S) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies

Access Microbiology ◽

10.1099/acmi.0.000057 ◽

2019 ◽

Vol 1 (9) ◽

Cited By ~ 5

Author(s):

Sandra Lester ◽

Jennifer Harcourt ◽

Michael Whitt ◽

Hail M. Al-Abdely ◽

Claire M. Midgley ◽

...

Keyword(s):

Middle East ◽

Vesicular Stomatitis Virus ◽

Neutralization Assay ◽

S Protein ◽

Vesicular Stomatitis ◽

Respiratory Coronavirus

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Homogeneous Surrogate Virus Neutralization Assay to Rapidly Assess Neutralization Activity of Anti-SARS-CoV-2 Antibodies

10.21203/rs.3.rs-1105720/v1 ◽

2021 ◽

Author(s):

Sun Jin Kim ◽

Zhong Yao ◽

Morgan Marsh ◽

Debra Eckert ◽

Michael Kay ◽

...

Keyword(s):

Immune Response ◽

Rapid Assessment ◽

Neutralization Assay ◽

Virus Neutralization ◽

Diagnostic Tools ◽

Neutralization Activity ◽

S Protein ◽

Serological Assay ◽

Virus Neutralization Assay ◽

Duration Of Protection

Abstract The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a ‘Serological Assay based on a Tri-part split-NanoLuc® (SATiN)’ to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Herein, we expand on our previous work and describe a reconfigured version of the SATiN assay that can measure neutralization activity of antibodies directly from convalescent or vaccinated sera. The sensitivity is comparable to cell-based pseudovirus neutralization assays but with significantly shorter preparation and assay run time. As the assay is modular, we further demonstrate that Neutralization SATiN (Neu-SATiN) enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

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Inhibitor screening of Spike variants reveals the heterogeneity of neutralizing antibodies to COVID-19 infection and vaccination

10.1101/2021.05.15.21257254 ◽

2021 ◽

Author(s):

Xiaomei Zhang ◽

Mei Zheng ◽

Te Liang ◽

Haijian Zhou ◽

Hongye Wang ◽

...

Keyword(s):

Drug Development ◽

Protective Effect ◽

Neutralizing Antibodies ◽

High Titer ◽

Neutralization Assay ◽

High Antibody ◽

Wild Type ◽

Antibody Diversity ◽

S Protein ◽

Inhibitor Screening

Mutations of the coronavirus responsible for coronavirus disease 2019 (COVID-19) could impede drug development and reduce the efficacy of COVID-19 vaccines. Here, we developed a multiplexed Spike-ACE2 Inhibitor Screening (mSAIS) assay that can measure the neutralizing effect of antibodies across numerous variants of the coronavirus's Spike (S) protein simultaneously. By screening purified antibodies and serum from convalescent COVID-19 patients and vaccinees against 72 S variants with the mSAIS assay, we identified new S mutations that are sensitive and resistant to neutralization. Serum from both infected and vaccinated groups with a high titer of neutralizing antibodies (NAbs) displayed a broader capacity to neutralize S variants than serum with low titer NAbs. These data were validated using serum from a large vaccinated cohort (n=104) with a tiled S peptide microarray. In addition, similar results were obtained using a SARS-CoV-2 pseudovirus neutralization assay specific for wild-type S and four prevalent S variants (D614G, B.1.1.7, B.1.351, P.1), thus demonstrating that high antibody diversity is associated with high NAb titers. Our results demonstrate the utility of the mSAIS platform in screening NAbs. Moreover, we show that heterogeneous antibody populations provide a more protective effect against S variants, which may help direct COVID-19 vaccine and drug development.

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MiR-210 and miR-155 are Upregulated in CFBE Cells and Involved in Fe-S Protein Assembly via ISCU Downregulation as well as HO-1 Expression via BACH1.

Pneumologie ◽

10.1055/s-0035-1556596 ◽

2015 ◽

Vol 69 (07) ◽

Author(s):

S Chillappagari ◽

V Garapati ◽

P Mahavadi ◽

O Stehling ◽

R Lill ◽

...

Keyword(s):

Protein Assembly ◽

S Protein

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A Hexagonal (II) Phase Phospholipid Neutralization Assay for Lupus Anticoagulant Identification

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649671 ◽

1993 ◽

Vol 70 (05) ◽

pp. 787-793 ◽

Cited By ~ 77

Author(s):

Douglas A Triplett ◽

Linda K Barna ◽

Gail A Unger

Keyword(s):

Hemophilia A ◽

Clinical Laboratory ◽

Neutralization Assay ◽

Confirmatory Test ◽

Specific Factor ◽

Clinical Settings ◽

Drug Induced ◽

Variable Screening ◽

Routine Clinical Laboratory

SummaryLupus anticoagulants (LAs) are immunoglobulins (IgG, IgM, or both) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e.g. APTT, dilute PT, dilute Russell Viper Venom Time). These antibodies may be identified in a wide variety of clinical settings. With the exception of heparinized patient samples, the presence of LAs is often the most common cause of an unexplained APTT in a routine clinical laboratory. The diagnosis of LAs is difficult due to variable screening reagent sensitivity and intrinsic heterogeneity of LAs. Recently, Rauch and colleagues have shown human monoclonal hybridoma LAs were inhibited by hexagonal (II) phase PLs. In contrast, lamellar phase PLs had no effect. We have evaluated a new assay system, Staclot LA®, which utilizes a hexagonal (II) phase PL (egg phosphatidylethanolamine [EPE]) as a confirmatory test for LAs. Plasma samples from the following patient populations were studied: LA positive, heparinized, oral anticoagulated, hemophilia A and B, and specific factor inhibitors (factors V, VIII, IX). Unlike previous studies, the LA positive patients were a mixed population including: autoimmune diseases, drug-induced, and post-infection. Our findings confirm the specificity of hexagonal (II) phase PL neutralization of LAs.

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