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. 2022 Jan 7;21(1):274-288.
doi: 10.1021/acs.jproteome.1c00882. Epub 2021 Dec 8.

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire

Rafael D Melani  1 , Benjamin J Des Soye  1   2 , Jared O Kafader  1 , Eleonora Forte  2   3 , Michael Hollas  1 , Voislav Blagojevic  1 , Fernanda Negrão  1 , John P McGee  1 , Bryon Drown  1 , Cameron Lloyd-Jones  1 , Henrique S Seckler  1 , Jeannie M Camarillo  1 , Philip D Compton  1   4 , Richard D LeDuc  1 , Bryan Early  1 , Ryan T Fellers  1 , Byoung-Kyu Cho  2 , Basil Baby Mattamana  2 , Young Ah Goo  1   2 , Paul M Thomas  1   2 , Michelle K Ash  5 , Pavan P Bhimalli  5 , Lena Al-Harthi  5 , Beverly E Sha  6 , Jeffrey R Schneider  5 , Neil L Kelleher  1   2   7
Affiliations

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire

Rafael D Melani et al. J Proteome Res. .

Abstract

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.

Keywords: COVID-19; SARS-CoV-2; antibodies; individual ion mass spectrometry; proteomics; serology; top-down mass spectrometry.

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Conflict of interest statement

Conflict of interest

N.L.K., J.O.K., and P.D.C. report a conflict of interest with I2MS technology used to readout the Ig profiles, currently being commercialized by Thermo Fisher Scientific.

Figures

Figure 1.
Figure 1.. Ig-MS, a new platform for COVID-19 serology.
(A) Overview of sample preparation and readout. Antibodies against the RBD domain of the SARS-CoV-2 spike protein are enriched from plasma or serum of COVID-19 patients, vaccinated, and controls individuals using magnetic beads conjugated with RBD. The eluates are then reduced to obtain light (LC) and heavy chains (HC), which are analyzed by individual ion mass spectrometry (I2MS). (B) Western blot showing the enrichment of Ig targeting SARS-CoV-2 spike RBD from CS1. (C) Example data set and metrics obtained from Ig-MS of the plasma from a COVID-19 convalescent patient (COVID-19_3) (only LC is shown).
Figure 2.
Figure 2.. Ig-MS readouts on a COVID-19 cohort.
(A-D) LC/HC spectral region and (E-H) expansion of the relative LC region for (A/E and B/F, red) two hospitalized patients and (C/G, purple) one outpatient. The standard mAb (CR3022) (D/H, blue) that binds SARS-CoV-2-RBD was used as a positive control (highlighted with gray vertical bar). (I) Ion titer (IT) and (J) degree of clonality (DoC) values obtained for all three outpatients and seven hospitalized patients. Analyses were done in triplicate. (K) Correlation of IT and (L) DoC values from Ig-MS with RBD-binding by ELISA, bioluminescent in vitro diagnostic (Promega), and surrogate virus neutralization. Shown are the Pearson correlation coefficient (r) and p-value (p).
Figure 3.
Figure 3.. Ig-MS readouts from the vaccinated cohort.
LC spectra for (A) uninfected individuals before the vaccination (time 0), (B) vaccinated individuals at 20 days after the first shot (time 1), and (C) at 28 days after the second shot (time 2); spiked-in standard mAb CR3022 highlighted by a gray rectangle. (D) IT and (E) DoC values of Ig-MS for all time points. (F) Bioluminescent in vitro diagnostic (Promega) titer of anti-RBD antibodies. (G) t-SNE plot generated with IT and DoC values from both analyzed cohorts.
Figure 4.
Figure 4.. LC/Fd Ig-MS readouts on the COVID-19 cohort.
(A) Overview of sample preparation and readout for LC/Fd Ig-MS. Antibodies against RBD are enriched from the plasma/serum of COVID-19 patients or control uninfected individuals with magnetic beads conjugated with RBD protein. Before elution, RBD-specific Igs are digested with the IdeS enzyme to remove the Fc domain of the HC. The eluates are then reduced to liberate LC and Fd, which are analyzed by I2MS. (B-E) LC/Fd spectral region for (B/C, red) two hospitalized patients and (D, purple) one outpatient. The standard mAb (CR3022) (E, blue) that binds SARS-CoV-2-RBD was used as a positive control (highlighted with gray vertical bar). (F) Ion titer (IT) and (G) degree of clonality (DoC) values were obtained for three outpatients and seven hospitalized patients. Analyses were done in triplicate. (H) Correlation of IT and (I) DoC values with RBD-binding by ELISA, bioluminescent in vitro diagnostic (Promega), and surrogate virus neutralization. Shown are the Pearson correlation coefficient (r) and p-value (p).
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References

    1. Zhou P; Yang XL; Wang XG; Hu B; Zhang L; Zhang W; Si HR; Zhu Y; Li B; Huang CL; Chen HD; Chen J; Luo Y; Guo H; Jiang RD; Liu MQ; Chen Y; Shen XR; Wang X; Zheng XS; Zhao K; Chen QJ; Deng F; Liu LL; Yan B; Zhan FX; Wang YY; Xiao GF; Shi ZL, A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, 579, (7798), 270–273. - PMC - PubMed
    1. Zhou Y; Yang Q; Chi J; Dong B; Lv W; Shen L; Wang Y, Comorbidities and the risk of severe or fatal outcomes associated with coronavirus disease 2019: A systematic review and meta-analysis. Int J Infect Dis 2020, 99, 47–56. - PMC - PubMed
    1. Ahmad FB; Cisewski JA; Minino A; Anderson RN, Provisional Mortality Data - United States, 2020. MMWR Morb Mortal Wkly Rep 2021, 70, (14), 519–522. - PMC - PubMed
    1. Wang P; Nair MS; Liu L; Iketani S; Luo Y; Guo Y; Wang M; Yu J; Zhang B; Kwong PD; Graham BS; Mascola JR; Chang JY; Yin MT; Sobieszczyk M; Kyratsous CA; Shapiro L; Sheng Z; Huang Y; Ho DD, Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 2021, 593, 130–135. - PubMed
    1. Wu J; Liang B; Chen C; Wang H; Fang Y; Shen S; Yang X; Wang B; Chen L; Chen Q; Wu Y; Liu J; Yang X; Li W; Zhu B; Zhou W; Wang H; Li S; Lu S; Liu D; Li H; Krawczyk A; Lu M; Yang D; Deng F; Dittmer U; Trilling M; Zheng X, SARS-CoV-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic COVID-19. Nat Commun 2021, 12, (1), 1813. - PMC - PubMed

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