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PLoS One ; 17(7): e0272298, 2022.
Article in English | MEDLINE | ID: covidwho-1963049


Virus neutralization assays provide a means to quantitate functional antibody responses that block virus infection. These assays are instrumental in defining vaccine and therapeutic antibody potency, immune evasion by viral variants, and post-infection immunity. Here we describe the development, optimization and evaluation of a live virus microneutralization assay specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this assay, SARS-CoV-2 clinical isolates are pre-incubated with serial diluted antibody and added to Vero E6 cells. Replicating virus is quantitated by enzyme-linked immunosorbent assay (ELISA) targeting the SARS-CoV-2 nucleocapsid protein and the standardized 50% virus inhibition titer calculated. We evaluated critical test parameters that include virus titration, assay linearity, number of cells, viral dose, incubation period post-inoculation, and normalization methods. Virus titration at 96 hours was determined optimal to account for different growth kinetics of clinical isolates. Nucleocapsid protein levels directly correlated with virus inoculum, with the strongest correlation at 24 hours post-inoculation. Variance was minimized by infecting a cell monolayer, rather than a cell suspension. Neutralization titers modestly decreased with increasing numbers of Vero E6 cells and virus amount. Application of two different normalization models effectively reduced the intermediate precision coefficient of variance to <16.5%. The SARS-CoV-2 microneutralization assay described and evaluated here is based on the influenza virus microneutralization assay described by WHO, and are proposed as a standard assay for comparing neutralization investigations.

COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Enzyme-Linked Immunosorbent Assay , Humans , Neutralization Tests/methods , Nucleocapsid Proteins , Spike Glycoprotein, Coronavirus
NPJ Vaccines ; 6(1): 156, 2021 Dec 20.
Article in English | MEDLINE | ID: covidwho-1585846


New generation plasmid DNA vaccines may be a safe, fast and simple emergency vaccine platform for preparedness against emerging viral pathogens. Applying platform optimization strategies, we tested the pre-clinical immunogenicity and protective effect of a candidate DNA plasmid vaccine specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The DNA vaccine induced spike-specific binding IgG and neutralizing antibodies in mice, rabbits, and rhesus macaques together with robust Th1 dominant cellular responses in small animals. Intradermal and intramuscular needle-free administration of the DNA vaccine yielded comparable immune responses. In a vaccination-challenge study of rhesus macaques, the vaccine demonstrated protection from viral replication in the lungs following intranasal and intratracheal inoculation with SARS-CoV-2. In conclusion, the candidate plasmid DNA vaccine encoding the SARS-CoV-2 spike protein is immunogenic in different models and confers protection against lung infection in nonhuman primates. Further evaluation of this DNA vaccine candidate in clinical trials is warranted.

Front Microbiol ; 12: 698944, 2021.
Article in English | MEDLINE | ID: covidwho-1305659


In addition to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit to animals that include hamsters, cats, dogs, mink, ferrets, tigers, lions, cynomolgus macaques, rhesus macaques, and treeshrew. Among these, mink are particularly susceptible. Indeed, 10 countries in Europe and North America reported SARS-CoV-2 infection among mink on fur farms. In Denmark, SARS-CoV-2 spread rapidly among mink farms and spilled-over back into humans, acquiring mutations/deletions with unknown consequences for virulence and antigenicity. Here we describe a mink-associated SARS-CoV-2 variant (Cluster 5) characterized by 11 amino acid substitutions and four amino acid deletions relative to Wuhan-Hu-1. Temporal virus titration, together with genomic and subgenomic viral RNA quantitation, demonstrated a modest in vitro fitness attenuation of the Cluster 5 virus in the Vero-E6 cell line. Potential alterations in antigenicity conferred by amino acid changes in the spike protein that include three substitutions (Y453F, I692V, and M1229I) and a loss of two amino acid residues 69 and 70 (ΔH69/V70), were evaluated in a virus microneutralization assay. Compared to a reference strain, the Cluster 5 variant showed reduced neutralization in a proportion of convalescent human COVID-19 samples. The findings underscore the need for active surveillance SARS-CoV-2 infection and virus evolution in susceptible animal hosts.