Evaluating SARS-CoV-2 spike and nucleocapsid proteins as targets for antibody detection in severe and mild COVID-19 cases using a Luminex bead-based assay.

Large-scale serosurveillance of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) will only be possible if serological tests are sufficiently reliable, rapid and affordable. Many assays are either labour-intensive and require specialised facilities (e.g. virus neutralization assays), or are expensive with suboptimal specificity (e.g. commercial ELISAs and RDTs). Bead-based assays offer a cost-effective alternative and allow for multiplexing to test for antibodies against multiple antigens and against other pathogens. Here, we compare the performance of spike (S) and nucleocapsid (NP) antigens for the detection of SARS-CoV-2 specific IgG, IgM and IgA antibodies in a panel of sera that includes recent (up to six weeks after symptom onset, severe n = 44; and mild cases n = 52) and old infections (five months after symptom onset, mild n = 104), using a Luminex-bead based assay and comparison to a virus neutralization test. While we show that neutralizing antibody levels are significantly lower in mild than in severe cases, we demonstrate that a combination of the recombinant nucleocapsid protein (NP) and receptor-binding domain (RBD) results in highly specific (99 %) IgG antibody detection five months after infection in 96 % of cases. Although most severe Covid-19 cases developed a clear IgM and IgA response, titers fell below the detection threshold in more than 20 % of mild cases in our bead-based assay. In conclusion, our data supports the use of RBD and NP for the development of SARS-CoV-2 serological IgG bead-based assays.

Mariën J ，Ceulemans A ，Michiels J ，Heyndrickx L ，Kerkhof K ，Foque N ，Widdowson MA ，Mortgat L ，Duysburgh E ，Desombere I ，Jansens H ，Van Esbroeck M ，Ariën KK ... -  《-》

Anti-SARS-CoV-2 Immunoglobulin Isotypes, and Neutralization Activity Against Viral Variants, According to BNT162b2-Vaccination and Infection History.

To compare SARS-CoV-2 antigen-specific antibody production and plasma neutralizing capacity against B.1 wild-type-like strain, and Gamma/P.1 and Delta/B.1.617.2 variants-of-concern, in subjects with different Covid-19 disease and vaccination histories. Adult subjects were: 1) Unvaccinated/hospitalized for Covid-19; 2) Covid-19-recovered followed by one BNT162b2 vaccine dose; and 3) Covid-19-naïve/2-dose BNT162b2 vaccinated. Multiplex Luminex® immunoassays measured IgG, IgA, and IgM plasma levels against SARS-CoV-2 receptor-binding domain (RBD), spike-1 (S), and nucleocapsid proteins. Neutralizing activity was determined in Vero E6 cytopathic assays. Maximum anti-RBD IgG levels were similar in Covid-19‑recovered individuals 8‒10 days after single-dose vaccination and in Covid-19-naïve subjects 7 days after 2nd vaccine dosing; both groups had ≈2‑fold higher anti-RBD IgG levels than Unvaccinated/Covid-19 subjects tracked through 2 weeks post-symptom onset. Anti-S IgG expression patterns were similar to RBD within each group, but with lower signal strengths. Viral antigen-specific IgA and IgM levels were more variable than IgG patterns. Anti-nucleocapsid immunoglobulins were not detected in Covid-19-naïve subjects. Neutralizing activity against the B.1 strain, and Gamma/P.1 and Delta/B.1.617.2 variants, was highest in Covid‑19-recovered/single-dose vaccinated subjects; although neutralization against the Delta variant in this group was only 26% compared to B.1 neutralization, absolute anti-Delta titers suggested maintained protection. Neutralizing titers against the Gamma and Delta variants were 33‒77% and 26‒67%, respectively, versus neutralization against the B.1 strain (100%) in the three groups. These findings support SARS-CoV-2 mRNA vaccine usefulness regardless of Covid-19 history, and confirm remarkable protection provided by a single vaccine dose in people who have recovered from Covid-19.

Tarkowski M ，de Jager W ，Schiuma M ，Covizzi A ，Lai A ，Gabrieli A ，Corbellino M ，Bergna A ，Ventura CD ，Galli M ，Riva A ，Antinori S ... -  《Frontiers in Immunology》

A Highly Sensitive and Specific SARS-CoV-2 Spike- and Nucleoprotein-Based Fluorescent Multiplex Immunoassay (FMIA) to Measure IgG, IgA, and IgM Class Antibodies.

Solastie A ，Virta C ，Haveri A ，Ekström N ，Kantele A ，Miettinen S ，Lempainen J ，Jalkanen P ，Kakkola L ，Dub T ，Julkunen I ，Melin M ... -  《Microbiology Spectrum》

A Combination of N and S Antigens With IgA and IgG Measurement Strengthens the Accuracy of SARS-CoV-2 Serodiagnostics.

Primary diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is based on detection of virus RNA in nasopharyngeal swab samples. In addition, analysis of humoral immunity against SARS-CoV-2 has an important role in viral diagnostics and seroprevalence estimates. We developed and optimized an enzyme immunoassays (EIA) using SARS-CoV-2 nucleoprotein (N), S1 and receptor binding domain (RBD) of the viral spike protein, and N proteins from SARS, Middle East respiratory syndrome (MERS), and 4 low-pathogenic human CoVs. Neutralizing antibody activity was compared with SARS-CoV-2 IgG, IgA, and IgM EIA results. The sensitivity of EIA for detecting immune response in COVID-19 patients (n = 101) was 77% in the acute phase and 100% in the convalescent phase of SARS-CoV-2 infection when N and RBD were used as antigens in IgG and IgA specific EIAs. SARS-CoV-2 infection significantly increased humoral immune responses against the 229E and NL63 N proteins. S1 and RBD-based EIA results had a strong correlation with microneutralization test results. The data indicate a combination of SARS-CoV-2 S1 or RBD and N proteins and analysis of IgG and IgA immunoglobulin classes in sera provide an excellent basis for specific and sensitive serological diagnostics of COVID-19.

Jalkanen P ，Pasternack A ，Maljanen S ，Melén K ，Kolehmainen P ，Huttunen M ，Lundberg R ，Tripathi L ，Khan H ，Ritvos MA ，Naves R ，Haveri A ，Österlund P ，Kuivanen S ，Jääskeläinen AJ ，Kurkela S ，Lappalainen M ，Rantasärkkä K ，Vuorinen T ，Hytönen J ，Waris M ，Tauriainen S ，Ritvos O ，Kakkola L ，Julkunen I ... -  《-》

A highly specific and sensitive serological assay detects SARS-CoV-2 antibody levels in COVID-19 patients that correlate with neutralization.

Peterhoff D ，Glück V ，Vogel M ，Schuster P ，Schütz A ，Neubert P ，Albert V ，Frisch S ，Kiessling M ，Pervan P ，Werner M ，Ritter N ，Babl L ，Deichner M ，Hanses F ，Lubnow M ，Müller T ，Lunz D ，Hitzenbichler F ，Audebert F ，Hähnel V ，Offner R ，Müller M ，Schmid S ，Burkhardt R ，Glück T ，Koller M ，Niller HH ，Graf B ，Salzberger B ，Wenzel JJ ，Jantsch J ，Gessner A ，Schmidt B ，Wagner R ... -  《-》