https://www.arca.fiocruz.br/bitstream/icict/47927/2/Reduced%20neutralization%20of%20SARS-CoV-2%20B.1.617%20by%20vaccine%20and%20convalescent%20serum.pdf

Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.

http://www.cell.com/article/S0092867422007103/pdf

Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum

Neutralisation assays are crucial tools to quantify the presence of functional neutralising antibodies in serum samples. Since the SARS-CoV-2 virus (the causative agent of COVID-19) is designated as a category 3 biosafety level pathogen, pseudotyped viruses bearing the SARS-CoV-2 spike protein permit extensive and widespread serum/plasma screening in a BSL 2 laboratory. These assays can be used to assess viral tropism, vaccine immunogenicity, efficacy of antiviral compounds (incl. therapeutic mAbs) and serosurveillance studies. In this article, we highlight approaches to SARS-CoV-2 viral pseudotyping, its practicality, and utility in increasing our understanding of neutralising antibodies against SARS-CoV-2.

The role of pseudotype neutralization assays in understanding SARS CoV-2

Understanding the levels of immunity in both the immunocompromised and general populations will facilitate the development of public health strategies to contain and prevent novel variants of interest or the emergence of vaccine-resistant variants that could threaten COVID-19 immunisation programmes.

Paucity and discordance of neutralising antibody responses to SARS-CoV-2 VOCs in vaccinated immunodeficient patients and health-care workers in the UK.

SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced 'COVID-19 like-illness', and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection.

https://www.mdpi.com/1999-4915/13/4/713/pdf

Comparison of Serological Assays for the Detection of SARS-CoV-2 Antibodies.

This protocol details a rapid and reliable method for the production and titration of high-titer viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G variant) on a lentiviral vector core, and use for neutralization assays in target cells expressing ACE2 and TMPRSS2. It additionally provides detailed instruction on substituting in new spike variants via gene cloning, and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titers to SARS-CoV and MERS-CoV pseudotypes, that they can be neutralized by human convalescent plasma and monoclonal antibodies and that they can be stored at a range of laboratory temperatures and lyophilized for distribution.

Production, titration, neutralisation and storage of SARS-CoV-2 lentiviral pseudotypes

Rationale: Exhaled breath condensate (EBC) promises a valuable, non-invasive, and easy to obtain clinical sample. However, it is not currently used diagnostically due to poor reproducibility, sample contamination, and sample loss. Objective: We evaluated whether a new, hand-held EBC collector (PBM-HALETM) that separates inertially impacted large droplets (LD) before condensing the fine aerosol (FA) fraction, in distinct self-sealing containers, overcomes current limitations. Methods: Sampling consistency was determined in healthy volunteers by microbial culture, 16S phylogenetics, spectrophotometry, RT-PCR, and HILIC-MS. Capture of aerosolised polystyrene beads, liposomes, virus-like particles, or reporter pseudotyped virus was analysed by nanoparticle tracking analysis, reporter expression assays, and flow cytometry. Acute symptomatic COVID-19 case tidal FA EBC viral load was quantified by RT-qPCR. Exhaled particles were counted by laser light scattering. Measurements and Main Results: Salivary amylase-free FA EBC capture was linear (R2=0.9992; 0.25-30 min) yielding RNA (6.03 g/mL) containing eukaryotic 18S rRNA (RT-qPCR; p<0.001) but not human GAPDH or beta actin mRNA, and 141 non-volatile metabolites including eukaryotic cell membrane components, and cuscohygrine 3 days after cocaine abuse. Culturable aerobe viability was condensation temperature-dependent. Breath fraction-specific microbiota were stable, identifying Streptococcus enrichment in a mild dry cough case. Nebulized pseudotyped virus infectivity loss <67% depended on condensation temperature, and particle charge-driven aggregation. No SARS-CoV-2 genomes were detected in convalescent or acute COVID-19 patient tidal breath FA EBC.

Airborne Pathogen Detection in Fine Aerosol Exhaled Oral Breath Condensates

The Eidolon helvum fruit bat is the most widely distributed fruit bat in Africa and is known to be a reservoir for several pathogenic viruses that can cause disease in humans. To assess the risk of zoonotic spillover, we conducted a serological survey of 304 serum samples from E. helvum bats that were captured for human consumption in Makurdi, Nigeria. Using pseudotyped viruses, we screened the samples for neutralising antibodies against viruses from the Coronaviridae, Filoviridae, Orthomyxoviridae and Paramyxoviridae families. We report the presence of neutralising antibodies against henipavirus lineage GH-M74a virus (odds ratio 6.23; p<0.001), Nipah virus (odds ratio 4.04; p=0.00031), bat influenza H17N10 virus (odds ratio 7.25; p<0.001) and no significant association with Ebola virus (odds ratio 0.56; p=0.375) in the bat cohort. The data suggest a potential risk of zoonotic spillover including the possible circulation of highly pathogenic viruses in E. helvum populations. These findings highlight the importance of maintaining sero-surveillance of E. helvum to monitor changes in virus prevalence and distribution over time and across different geographic locations. Article summary line The detection of neutralizing antibodies against henipavirus GH-M74a virus, Nipah virus, and H17N10 virus in Eidolon helvum bat sera from Nigeria using pseudotyped viruses suggests a potential risk of zoonotic spillover.

/pdf/serological-evidence-of-high-pathogenicity-virus-infection-1difn8ri.pdf

Bethany Auld

Papers

Production, titration, neutralisation and storage of SARS-CoV-2 lentiviral pseudotypes

Airborne Pathogen Detection in Fine Aerosol Exhaled Oral Breath Condensates

Serological evidence of high pathogenicity virus infection in Eidolon helvum fruit bats in Nigeria

Evaluation of Bioinformatically Designed, Historical Glycoproteins to Aid Pan-Lyssavirus Vaccine Development