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Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19

Nature Medicine volume 26, pages 1428–1434 (2020)Cite this article

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts1,2,3, most targeting the viral ‘spike’ glycoprotein (S). S localizes on the virion surface and mediates recognition of cellular receptor angiotensin-converting enzyme 2 (ACE2)4,5,6. Eliciting neutralizing antibodies that block S–ACE2 interaction7,8,9, or indirectly prevent membrane fusion10, constitute an attractive modality for vaccine-elicited protection11. However, although prototypic S-based vaccines show promise in animal models12,13,14, the immunogenic properties of S in humans are poorly resolved. In this study, we characterized humoral and circulating follicular helper T cell (cTFH) immunity against spike in recovered patients with coronavirus disease 2019 (COVID-19). We found that S-specific antibodies, memory B cells and cTFH are consistently elicited after SARS-CoV-2 infection, demarking robust humoral immunity and positively associated with plasma neutralizing activity. Comparatively low frequencies of B cells or cTFH specific for the receptor binding domain of S were elicited. Notably, the phenotype of S-specific cTFH differentiated subjects with potent neutralizing responses, providing a potential biomarker of potency for S-based vaccines entering the clinic. Overall, although patients who recovered from COVID-19 displayed multiple hallmarks of effective immune recognition of S, the wide spectrum of neutralizing activity observed suggests that vaccines might require strategies to selectively target the most potent neutralizing epitopes.

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Fig. 1: Serological responses to COVID-19.
Fig. 2: Frequency and phenotype of SARS-CoV-2-specific B cells after infection.
Fig. 3: Specificity of cTFH responses to coronavirus spike proteins.
Fig. 4: Predictors of plasma neutralization activity.

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Data availability

The mass spectrometry data that support the findings of this study are available via ProteomeXchange with identifier PXD019163. The authors declare that all other data supporting the findings of this study are available in the paper and the Supplementary Information files.

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Acknowledgements

We thank the generous participation of the trial individuals for providing samples. The SARS-CoV-2 RBD expression plasmids were kindly provided by F. Krammer (Icahn School of Medicine at Mt. Sinai). The human and mouse ACE2 expression plasmids were kindly provided by M. Thomas (Monash University). We acknowledge V. Jameson and staff at the Melbourne Cytometry Platform (Melbourne Brain Centre node) for provision of flow cytometry services. We thank the Melbourne Mass Spectrometry and Proteomics Facility of the Bio21 Molecular Science and Biotechnology Institute at the University of Melbourne for the support of mass spectrometry analysis. This study was supported by the Victorian Government and Medical Research Future Fund (MRFF) GNT2002073 (to W.-H.T., D.I.G., M.P.D., S.J.K. and A.K.W.), the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology (to S.J.K.), National Health and Medical Research Council (NHMRC) program grant APP1149990 (to S.J.K. and M.P.D.), NHMRC project grant GNT1162760 (to A.K.W), the Jack Ma Foundation (to D.I.G., N.A.G. and K.S.) and the A2 Milk Company (to K.S.). W-H.T. is a Howard Hughes Medical Institute–Wellcome Trust International Research Scholar (208693/Z/17/Z). J.A.J., D.I.G., M.P.D., W.-H.T., S.J.K. and A.K.W. are supported by NHMRC fellowships. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.

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Author notes

  1. These authors contributed equally: Jennifer A. Juno, Hyon-Xhi Tan, Stephen J. Kent, Adam K. Wheatley.

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

    Jennifer A. Juno, Hyon-Xhi Tan, Wen Shi Lee, Hannah G. Kelly, Kathleen Wragg, Robyn Esterbauer, Helen E. Kent, C. Jane Batten, Francesca L. Mordant, Nicholas A. Gherardin, Nichollas E. Scott, Dale I. Godfrey, Kanta Subbarao, Stephen J. Kent & Adam K. Wheatley

  2. Kirby Institute, University of New South Wales, Kensington, New South Wales, Australia

    Arnold Reynaldi & Miles P. Davenport

  3. Australian Research Council Centre for Excellence in Convergent Bio-Nano Science and Technology, University of Melbourne, Melbourne, Victoria, Australia

    Hannah G. Kelly, Robyn Esterbauer, Stephen J. Kent & Adam K. Wheatley

  4. Melbourne Sexual Health Centre and Department of Infectious Diseases, Alfred Hospital and Central Clinical School, Monash University, Melbourne, Victoria, Australia

    Helen E. Kent & Stephen J. Kent

  5. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Melbourne, Victoria, Australia

    Nicholas A. Gherardin & Dale I. Godfrey

  6. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Phillip Pymm, Melanie H. Dietrich & Wai-Hong Tham

  7. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia

    Phillip Pymm & Melanie H. Dietrich

  8. WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

    Kanta Subbarao

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Contributions

J.A.J., H.-X.T., W.S.L., S.J.K. and A.K.W. designed the study and experiments. J.A.J., H.-X.T., W.S.L., A.R., H.G.K., K.W., R.E., H.E.K, C.J.B., F.L.M., N.A.G., P.P., M.H.D., N.E.S. and A.K.W. performed experiments. W.-H.T., N.A.G., D.I.G. and K.S. contributed unique reagents. J.A.J., H.-X.T., W.S.L., A.R., K.S., M.P.D., S.J.K. and A.K.W. analyzed the experimental data. J.A.J., H.-X.T., W.S.L., A.R., M.P.D., S.J.K. and A.K.W. wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Stephen J. Kent or Adam K. Wheatley.

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The authors declare no competing interests.

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Peer review information Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1

Demographics and clinical characteristics of recruited COVID-19 patients and healthy control subjects.

Extended Data Fig. 2 Pre-screen of HCoV-HKU1 serum endpoint titres among a cohort of healthy subjects (N = 27) bled prior to the SARS-CoV-2 pandemic.

Data is shown as the median.

Extended Data Fig. 3 Correlations between antibody binding titres, ACE2/RBD binding inhibition and neutralization activity in plasma from subjects recovered from SARS-CoV-2 infection.

a, Spearman’s Correlation between endpoint titres of S-specific plasma antibody and the extent of ACE2/RBD binding inhibition, plasma neutralization titres or endpoint titres of RBD-specific plasma antibody (N = 41). b, Correlation between endpoint titres of RBD-specific plasma antibody and the extent of ACE2/RBD binding inhibition or plasma neutralziation activity (N = 41). c, Correlation between plasma ACE2/RBD binding inhibition and neutralization activity (N = 41). All correlations are two-tailed.

Extended Data Fig. 4 Representative staining of S- and RBD-specific IgD-IgG+ B cells.

3 uninfected subjects (left panels) and 6 subjects after recovery from SARS-CoV-2 infection (middle and right panels). CD19+IgD-IgG+ B cells cells were identified using gating strategy shown in Supplementary Fig. 1. Binding to SARS-CoV-2 spike and/or SARS-CoV-2 RBD probes was assessed.

Extended Data Fig. 5 Memory B cell phenotypes in subjects after SARS-CoV-2 infection.

a, b, Representative memory B cell phenotypes identified by CD21 and CD27 co-stain of probe+CD19+IgD- cells (blue) overlaid on CD19+IgD- cells (grey) (a) and the corresponding frequencies of the four populations in subjects previously infected with SARS-CoV-2 (b) (Resting memory—CD21+CD27+; activated memory—CD21−CD27+; naïve/CD27lo memory—CD21+CD27−; atypical B cells—CD21−CD27−); n.d—not detected due to absent probe+ cells. Memory B cell phenotypes were identified using gating strategy shown in Supplementary Fig. 2.

Extended Data Fig. 6 Analysis of V gene distribution, somatic mutation and CDR-H3 lengths for S- and RBD-specific BCR sequences.

a,b,c, Frequency distributions of human IGHV (a), IGKV (b) and IGLV (c) genes from BCR sequences recovered from S- (red) and RBD-binding (blue) IgG+ B cells from recovered COVID-19 patients (N = 5) with reference to distribution in cord blood (grey)49. Few IGLV sequences for RBD-binding B cells were recovered and are not shown. d, e, CDR-H3 (d) and somatic mutation (e) in S- (red) and RBD-binding (blue) IgG+ B cells compared to influenza B HA-specific IgG+ B cells recovered after immunisation23. f, Somatic mutation in S- (red) and RBD-binding (blue) IgG+ B cells for each individual subject. d,e,f, Data are shown as the median.

Extended Data Fig. 7 cTFH populations in SARS-CoV-2 positive and negative donors.

a, cTFH frequency (as a proportion of CD4+CD45RA– cells) among SARS-CoV-2 negative (n = 10) and SARS-CoV-2 positive (n = 39) donors. b, Frequency of antigen-specific cTFH following stimulation with peptide pools spanning S1 (without RBD peptides), S2 or RBD regions of SARS-CoV-2 S in SARS-CoV-2 positive donors (n = 20). c, Frequency of IFNγ+, IL-17+ or TNF+ cells following SEB stimulation among cTFH or Tmem (CD4+CD45RA–CXCR5−) populations in SARS-CoV-2 positive donors (n = 20; Wilcoxon two-tailed test). d, CCR6 and CXCR3 expression among bulk cTFH (grey), IFNγ+ (blue) or IL-17+ (red) cTFH cells following SEB stimulation in SARS-CoV-2 positive donors (n = 20). a, b, d, Data are shown as the median with interquartile range.

Extended Data Fig. 8 Peptide-specific cTFH responses in SARS-CoV-2 positive donors.

a, A 15-mer peptide derived from SARS-CoV-2 S2, LLQYGSFCTQLNRAL, was found to be immunogenic in 3 SARS-CoV-2 positive subjects (CP39, CP02, CP36). CP12 shown as representative non-responder. b, Plots indicate the CCR6/CXCR3 expression among S2 peptide pool- or peptide-specific cTFH for each donor.

Extended Data Fig. 9 CD4+ Tmem responses in COVID patients and controls.

a, Representative staining of CD134 and CD25 expression following protein or SEB stimulation among CD4 Tmem (CD4+CD45RA-CXCR5-) cells. b, Background-subtracted frequencies of SARS-CoV-2 S, SARS-CoV-2 RBD or HCoV HKU1 spike-specific CD4 Tmem cells among SARS-CoV-2 negative (n = 10) or SARS-CoV-2 positive (n = 39) donors. c, IFNγ, IL-17 and TNF responses following stimulation with overlapping peptide pools covering SARS-CoV-2 S1 or S2 domains, or SEB in SARS-CoV-2 positive donors (n = 20). d, Graphs indicate background-subtracted responses; responses not above background are indicated as 0.001% (n = 20). b, d, Data are shown as the median with interquartile range.

Extended Data Fig. 10 Association of immunological features with disease severity.

a, Analysis of the relationship between disease severity (mild, moderate or severe) and immunological parameters within the SARS-CoV-2 convalescent cohort. p < 0.05 indicated in bold (Kruskal-Wallis test, two-tailed). b, Relationship between cTFH, S-specific antibody titre, or microneutralization (MN) titre with disease severity (n = 26 mild, n = 10 moderate, n = 5 severe). Data are shown as the median with interquartile range.

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Juno, J.A., Tan, HX., Lee, W.S. et al. Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19. Nat Med 26, 1428–1434 (2020). https://doi.org/10.1038/s41591-020-0995-0

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