Comparability of six different immunoassays measuring SARS-CoV-2 antibodies with neutralizing antibody levels in convalescent plasma: From utility to prediction.

BACKGROUND: Convalescent plasma (CP) therapy for coronavirus disease (COVID-19) provides virus-neutralizing antibodies that may ameliorate the outcome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. The effectiveness of CP likely depends on its antiviral neutralizing potency and is determined using in vitro neutralizing antibody assays. STUDY DESIGN AND METHODS: We evaluated abilities of three immunoassays for anti-spike antibodies (EUROimmun, Ortho, Roche), a pseudotype-based neutralization assay, and two assays that quantify ACE2 binding of spike protein (GenScript and hemagglutination test [HAT]-based assay) to predict neutralizing antibody titers in 113 CP donations. Assay outputs were analyzed through linear regression and calculation of sensitivities and specificities by receiver operator characteristic (ROC) analysis. RESULTS: Median values of plasma samples containing neutralizing antibodies produced conversion factors for assay unitage of ×6.5 (pseudotype), ×19 (GenScript), ×3.4 (HAT assay), ×0.08 (EUROimmun), ×1.64 (Roche), and ×0.10 (Ortho). All selected assays were sufficient in identifying the high titer donations based on ROC analysis; area over curve ranged from 91.7% for HAT and GenScript assay to 95.6% for pseudotype assay. However, their ability to predict the actual neutralizing antibody levels varied substantially as shown by linear regression correlation values (from 0.27 for Ortho to 0.61 for pseudotype assay). DISCUSSION: Overall, the study data demonstrate that all selected assays were effective in identifying donations with high neutralizing antibody levels and are potentially suitable as surrogate assays for donation selection for CP therapy.

Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein.

We have produced a new Ebola virus pseudotype, E-S-FLU, that can be handled in biosafety level 1/2 containment for laboratory analysis. The E-S-FLU virus is a single-cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza virus hemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU virus production. Infection of cells with the E-S-FLU virus was dependent on the Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. The E-S-FLU virus was neutralized specifically by an anti-Ebolavirus glycoprotein antibody and a variety of small drug molecules that are known to inhibit the entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC1280; Sigma) of 1,280 pharmacologically active compounds for inhibition of virus entry. A total of 215 compounds inhibited E-S-FLU virus infection, while only 22 inhibited the control H5-S-FLU virus coated in H5 hemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action, e.g., calcium channel blockers, estrogen receptor antagonists, antihistamines, serotonin uptake inhibitors, etc., and this correlates with inhibitor screening results obtained with other pseudotypes or wild-type Ebola virus in the literature. The E-S-FLU virus is a new tool for Ebola virus cell entry studies and is easily applied to high-throughput screening assays for small-molecule inhibitors or antibodies.IMPORTANCE Ebola virus is in the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates for Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus and screen for inhibitors. Here we characterized a new surrogate, named E-S-FLU virus, that is based on a disabled influenza virus core coated with the Ebola virus surface protein but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU virus uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU virus in biosafety level 1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small-molecule library of 1,280 candidates for inhibitors of viral entry.

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions.

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.