Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome
<p>SARS-CoV-2 nucleocapsid and spike proteins elicit a robust IgA, total IgG and IgG-subclass response in COVID-19 patients. (<b>A</b>) Sketch depicts SARS-CoV-2 full-length and truncated nucleocapsid (N) and spike (S) proteins. (<b>B</b>) RT-PCR confirmed COVID-19 patients (n = 128) with non-severe disease (outpatient n = 80) and hospitalized patients with severe disease (inpatient n = 48) included in the study. (<b>C</b>–<b>E</b>) 100 ng of purified SARS-CoV-2 proteins were coated on ELISA plates and 1:100 patient serum dilution was used in an indirect ELISA to estimate serum IgA, IgG and IgG-subclass response. Antigen-specific antibody response among (<b>C</b>) all COVID-19 patients, (<b>D</b>) outpatients and inpatients, and (<b>E</b>) discharged (n = 24, samples n = 59) and deceased (n = 24, samples n = 72) inpatients was compared. Reactivity index (RI) values were calculated as a ratio between sample optical density and pre-pandemic negative samples. Horizontal red or black lines denote the median antibody range. Each curve in the graph represents reactivity towards each antigen, Splines were plotted using the Fit Spline program in GraphPad Prism software. ANOVA test with Tukey’s post hoc test was used to compare differences in the RI between antigens. Two-way ANOVA with Šidák post hoc correction for multiple comparisons. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 2
<p>Representative COVID-19 patients and their humoral response: 100 ng of purified SARS-CoV-2 protein and 1:100 patient serum dilution was used in an indirect ELISA to estimate serum IgA, total IgG, IgG-subclass response and RBD-ACE inhibitory antibodies. Each column represents one patient, and each row represents specific response to one antibody isotype or RBD-ACE2 inhibitory antibodies. Each graph has reactivity index for SARS-CoV-2 full-length or truncated antigens tested. Outpatient samples were collected only at recruitment, whereas inpatients were followed longitudinally after hospitalization. Vertical dotted blank and red lines indicate hospitalized and intubation period, respectively (samples n = 3).</p> "> Figure 3
<p>RBD-ACE2 interactions blocking antibodies and neutralizing antibody increase with symptomatic days and correlates with disease severity. (<b>A</b>) Commercial RBD-ACE2 competitive assay was used to assess inhibitory antibodies among COVID-19 patients (n = 111, samples n = 190) versus healthy individuals (n = 13). (<b>B</b>–<b>D</b>) Percentage RBD-ACE2 inhibition was evaluated post-onset of symptoms (outpatients n = 70; discharged inpatients n = 20, samples n = 54; deceased inpatients n = 21, samples n = 66). (<b>E</b>–<b>H</b>) Plasma samples were tested by PRNT in Vero cells after incubation with 300 plaque forming units (PFU) (outpatients n = 40; discharged inpatients n = 6, samples n = 18; deceased inpatients n = 8, samples n = 24). (<b>E</b>) Correlation between RBD-ACE2 competitive assay and live-virus PRNT assay (samples n = 82). (<b>F</b>) Outpatient and inpatient patient samples were serially diluted for the PRNT assay. Each data point represents the mean of all plasma samples for each group at each dilution level and error bars represent SD. (<b>G</b>,<b>H</b>) Percentage neutralization values were compared to days after onset of symptoms to evaluate neutralizing antibody kinetics. Solid lines representing the tendency for the neutralizing antibodies were created by the Fit Spline program in GraphPad Prism software. Two-way ANOVA with Šidák post hoc correction for multiple comparisons was applied. **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 4
<p>Elevated antigen-specific IgG3 response correlated with severe disease outcome. IgG subclass antibody ratio correlates with disease outcome. Ratio of full-length and truncated N and S protein-specific IgG subclass antibody response was compared between (<b>A</b>) outpatients (n = 45) and inpatients (n = 38, samples n = 121) or (<b>B</b>) hospitalized patients with discharged (n = 20, samples n = 55) or deceased (n = 18, samples n = 66) disease outcome. Samples with more than seven days after the start of symptoms were included in this analysis. T-test was performed to compare patient groups. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics
2.2. Patient Recruitment and Sampling
2.3. SARS-CoV-2 Proteins
2.4. Measurement of IgA, IgG and IgG-Subclass Response
2.5. RBD-ACE2 Competitive Assay
2.6. SARS-CoV-2 Plaque Reduction Neutralization Test
2.7. Statistics and Data Analysis
3. Results
3.1. Patient Demographics
3.2. Robust Humoral Response against the Nucleocapsid and Spike Full-Length, and Truncated Proteins
3.3. Presence of SARS-CoV-2 Neutralizing Antibodies Does Not Affect Disease Outcome
3.4. Relationship between IgG Subclass Signature and COVID-19 Outcome
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Brizzi, A.; Whittaker, C.; Servo, L.M.S.; Hawryluk, I.; Prete, C.A.; de Souza, W.M.; Aguiar, R.S.; Araujo, L.J.T.; Bastos, L.S.; Blenkinsop, A.; et al. Spatial and temporal fluctuations in COVID-19 fatality rates in Brazilian hospitals. Nat. Med. 2022, 28, 1476–1485. [Google Scholar] [CrossRef] [PubMed]
- Earle, K.A.; Ambrosino, D.M.; Fiore-Gartland, A.; Goldblatt, D.; Gilbert, P.B.; Siber, G.R.; Dull, P.; Plotkin, S.A. Evidence for antibody as a protective correlate for COVID-19 vaccines. Vaccine 2021, 39, 4423–4428. [Google Scholar] [CrossRef] [PubMed]
- Wu, F.; Zhao, S.; Yu, B.; Chen, Y.-M.; Wang, W.; Song, Z.-G.; Hu, Y.; Tao, Z.-W.; Tian, J.-H.; Pei, Y.-Y.; et al. A new coronavirus associated with human respiratory disease in China. Nature 2020, 579, 265–269. [Google Scholar] [CrossRef] [PubMed]
- Lu, R.; Zhao, X.; Li, J.; Niu, P.; Yang, B.; Wu, H.; Wang, W.; Song, H.; Huang, B.; Zhu, N.; et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet 2020, 395, 565–574. [Google Scholar] [CrossRef]
- V’Kovski, P.; Kratzel, A.; Steiner, S.; Stalder, H.; Thiel, V. Coronavirus biology and replication: Implications for SARS-CoV-2. Nat. Rev. Microbiol. 2021, 19, 155–170. [Google Scholar] [CrossRef]
- Bojkova, D.; Klann, K.; Koch, B.; Widera, M.; Krause, D.; Ciesek, S.; Cinatl, J.; Münch, C. Proteomics of SARS-CoV-2-infected host cells reveals therapy targets. Nature 2020, 583, 469–472. [Google Scholar] [CrossRef]
- Jayaram, J.; Youn, S.; Collisson, E.W. The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates. Virology 2005, 339, 127–135. [Google Scholar] [CrossRef]
- Poland, G.A.; Ovsyannikova, I.G.; Kennedy, R.B. SARS-CoV-2 immunity: Review and applications to phase 3 vaccine candidates. Lancet 2020, 396, 1595–1606. [Google Scholar] [CrossRef]
- Deeks, J.J.; Dinnes, J.; Takwoingi, Y.; Davenport, C.; Spijker, R.; Taylor-Phillips, S.; Adriano, A.; Beese, S.; Dretzke, J.; di Ruffano, L.F.; et al. Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane Database Syst. Rev. 2020, 6, CD013652. [Google Scholar] [CrossRef]
- Fazolo, T.; Lima, K.; Fontoura, J.C.; de Souza, P.O.; Hilario, G.; Zorzetto, R.; Júnior, L.R.; Pscheidt, V.M.; Neto, J.d.C.F.; Haubert, A.F.; et al. Pediatric COVID-19 patients in South Brazil show abundant viral mRNA and strong specific anti-viral responses. Nat. Commun. 2021, 12, 6844. [Google Scholar] [CrossRef]
- Borba, M.G.S.; Val, F.F.A.; Sampaio, V.S.; Alexandre, M.A.A.; Melo, G.C.; Brito, M.; Mourao, M.P.G.; Brito-Sousa, J.D.; Baia-da-Silva, D.; Guerra, M.V.F.; et al. Effect of High vs Low Doses of Chloroquine Diphosphate as Adjunctive Therapy for Patients Hospitalized With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: A Randomized Clinical Trial. JAMA Netw. Open 2020, 3, e208857. [Google Scholar] [CrossRef] [PubMed]
- Lalwani, P.; Salgado, B.B.; Pereira Filho, I.V.; Silva, D.S.S.d.; Morais, T.B.d.N.d.; Jordão, M.F.; Barbosa, A.R.C.; Cordeiro, I.B.; Neto, J.N.d.S.; Assunção, E.N.d.; et al. SARS-CoV-2 Seroprevalence and Associated Factors in Manaus, Brazil: Baseline Results from the DETECTCoV-19 Cohort Study. Int. J. Infect. Dis. 2021, 110, 141–150. [Google Scholar] [CrossRef] [PubMed]
- Alvim, R.G.F.; Lima, T.M.; Rodrigues, D.A.S.; Marsili, F.F.; Bozza, V.B.T.; Higa, L.M.; Monteiro, F.L.; Leitao, I.C.; Carvalho, R.S.; Galliez, R.M.; et al. From a recombinant key antigen to an accurate, affordable serological test: Lessons learnt from COVID-19 for future pandemics. Biochem. Eng. J. 2022, 186, 108537. [Google Scholar] [CrossRef] [PubMed]
- Souza, W.M.; Amorim, M.R.; Sesti-Costa, R.; Coimbra, L.D.; Brunetti, N.S.; Toledo-Teixeira, D.A.; de Souza, G.F.; Muraro, S.P.; Parise, P.L.; Barbosa, P.P.; et al. Neutralisation of SARS-CoV-2 lineage P.1 by antibodies elicited through natural SARS-CoV-2 infection or vaccination with an inactivated SARS-CoV-2 vaccine: An immunological study. Lancet Microbe 2021, 2, e527–e535. [Google Scholar] [CrossRef]
- Long, Q.-X.; Liu, B.-Z.; Deng, H.-J.; Wu, G.-C.; Deng, K.; Chen, Y.-K.; Liao, P.; Qiu, J.-F.; Lin, Y.; Cai, X.-F.; et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat. Med. 2020, 26, 845–848. [Google Scholar] [CrossRef]
- Guthmiller, J.J.; Stovicek, O.; Wang, J.; Changrob, S.; Li, L.; Halfmann, P.; Zheng, N.-Y.; Utset, H.; Stamper, C.T.; Dugan, H.L.; et al. SARS-CoV-2 Infection Severity Is Linked to Superior Humoral Immunity against the Spike. mBio 2021, 12, e02940-20. [Google Scholar] [CrossRef]
- Röltgen, K.; Powell, A.E.; Wirz, O.F.; Stevens, B.A.; Hogan, C.A.; Najeeb, J.; Hunter, M.; Wang, H.; Sahoo, M.K.; Huang, C.; et al. Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Sci. Immunol. 2020, 5, abe0240. [Google Scholar] [CrossRef]
- Okba, N.M.; Raj, V.S.; Widjaja, I.; GeurtsvanKessel, C.H.; de Bruin, E.; Chandler, F.D.; Park, W.B.; Kim, N.-J.; Farag, E.A.; Al-Hajri, M.; et al. Sensitive and Specific Detection of Low-Level Antibody Responses in Mild Middle East Respiratory Syndrome Coronavirus Infections. Emerg. Infect. Dis. 2019, 25, 1868–1877. [Google Scholar] [CrossRef]
- Alshukairi, A.N.; Khalid, I.; Ahmed, W.A.; Dada, A.M.; Bayumi, D.T.; Malic, L.S.; Althawadi, S.; Ignacio, K.; Alsalmi, H.S.; Al-Abdely, H.M.; et al. Antibody Response and Disease Severity in Healthcare Worker MERS Survivors. Emerg. Infect. Dis. 2016, 22, 1113–1115. [Google Scholar] [CrossRef]
- Drosten, C.; Meyer, B.; Müller, M.A.; Corman, V.M.; Al-Masri, M.; Hossain, R.; Madani, H.; Sieberg, A.; Bosch, B.J.; Lattwein, E.; et al. Transmission of MERS-Coronavirus in Household Contacts. N. Engl. J. Med. 2014, 371, 828–835. [Google Scholar] [CrossRef]
- Nilsson, A.C.; Holm, D.K.; Justesen, U.S.; Gorm-Jensen, T.; Andersen, N.S.; Øvrehus, A.; Johansen, I.S.; Michelsen, J.; Sprogøe, U.; Lillevang, S.T. Comparison of six commercially available SARS-CoV-2 antibody assays—Choice of assay depends on intended use. Int. J. Infect. Dis. 2020, 103, 381–388. [Google Scholar] [CrossRef] [PubMed]
- Anna, F.; Goyard, S.; Lalanne, A.I.; Nevo, F.; Gransagne, M.; Souque, P.; Louis, D.; Gillon, V.; Turbiez, I.; Bidard, F.C.; et al. High seroprevalence but short-lived immune response to SARS-CoV-2 infection in Paris. Eur. J. Immunol. 2021, 51, 180–190. [Google Scholar] [CrossRef] [PubMed]
- Moncunill, G.; Mayor, A.; Santano, R.; Jiménez, A.; Vidal, M.; Tortajada, M.; Sanz, S.; Méndez, S.; Llupià, A.; Aguilar, R.; et al. SARS-CoV-2 Seroprevalence and Antibody Kinetics Among Health Care Workers in a Spanish Hospital After 3 Months of Follow-up. J. Infect. Dis. 2020, 223, 62–71. [Google Scholar] [CrossRef] [PubMed]
- Dobaño, C.; Santano, R.; Jiménez, A.; Vidal, M.; Chi, J.; Melero, N.R.; Popovic, M.; López-Aladid, R.; Fernández-Barat, L.; Tortajada, M.; et al. Immunogenicity and crossreactivity of antibodies to the nucleocapsid protein of SARS-CoV-2: Utility and limitations in seroprevalence and immunity studies. Transl. Res. 2021, 232, 60–74. [Google Scholar] [CrossRef]
- Atyeo, C.; Fischinger, S.; Zohar, T.; Slein, M.D.; Burke, J.; Loos, C.; McCulloch, D.J.; Newman, K.L.; Wolf, C.; Yu, J.; et al. Distinct Early Serological Signatures Track with SARS-CoV-2 Survival. Immunity 2020, 53, 524–532.e4. [Google Scholar] [CrossRef]
- Iles, J.K.; Zmuidinaite, R.; Sadee, C.; Gardiner, A.; Lacey, J.; Harding, S.; Wallis, G.; Patel, R.; Roblett, D.; Heeney, J.; et al. Determination of IgG1 and IgG3 SARS-CoV-2 Spike Protein and Nucleocapsid Binding—Who Is Binding Who and Why? Int. J. Mol. Sci. 2022, 23, 6050. [Google Scholar] [CrossRef]
- Yates, J.L.; Ehrbar, D.J.; Hunt, D.T.; Girardin, R.C.; Dupuis, A.P.; Payne, A.F.; Sowizral, M.; Varney, S.; Kulas, K.E.; Demarest, V.L.; et al. Serological analysis reveals an imbalanced IgG subclass composition associated with COVID-19 disease severity. Cell Rep. Med. 2021, 2, 100329. [Google Scholar] [CrossRef]
- Liu, Z.; Long, W.; Tu, M.; Chen, S.; Huang, Y.; Wang, S.; Zhou, W.; Chen, D.; Zhou, L.; Wang, M.; et al. Lymphocyte subset (CD4+, CD8+) counts reflect the severity of infection and predict the clinical outcomes in patients with COVID-19. J. Infect. 2020, 81, 318–356. [Google Scholar] [CrossRef]
- Huang, W.; Berube, J.; McNamara, M.; Saksena, S.; Hartman, M.; Arshad, T.; Bornheimer, S.J.; O’Gorman, M. Lymphocyte Subset Counts in COVID-19 Patients: A Meta-Analysis. Cytom. Part A 2020, 97, 772–776. [Google Scholar] [CrossRef]
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Salgado, B.B.; Jordão, M.F.; de Morais, T.B.d.N.; da Silva, D.S.S.; Pereira Filho, I.V.; Salgado Sobrinho, W.B.; Carvalho, N.O.; dos Santos, R.O.; Forato, J.; Barbosa, P.P.; et al. Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome. Viruses 2023, 15, 1018. https://doi.org/10.3390/v15041018
Salgado BB, Jordão MF, de Morais TBdN, da Silva DSS, Pereira Filho IV, Salgado Sobrinho WB, Carvalho NO, dos Santos RO, Forato J, Barbosa PP, et al. Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome. Viruses. 2023; 15(4):1018. https://doi.org/10.3390/v15041018
Chicago/Turabian StyleSalgado, Bárbara Batista, Maele Ferreira Jordão, Thiago Barros do Nascimento de Morais, Danielle Severino Sena da Silva, Ivanildo Vieira Pereira Filho, Wlademir Braga Salgado Sobrinho, Nani Oliveira Carvalho, Rafaella Oliveira dos Santos, Julia Forato, Priscilla Paschoal Barbosa, and et al. 2023. "Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome" Viruses 15, no. 4: 1018. https://doi.org/10.3390/v15041018