Single-Cell Immunogenomic Approach Identified SARS-CoV-2 Protective Immune Signatures in Asymptomatic Direct Contacts of COVID-19 Cases

Abstract

The response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely impacted by the level of virus exposure and status of the host immunity. The nature of protection shown by direct asymptomatic contacts of coronavirus disease 2019 (COVID-19)-positive patients is quite intriguing. In this study, we have characterized the antibody titer, SARS-CoV-2 surrogate virus neutralization, cytokine levels, single-cell T-cell receptor (TCR), and B-cell receptor (BCR) profiling in asymptomatic direct contacts, infected cases, and controls. We observed significant increase in antibodies with neutralizing amplitude in asymptomatic contacts along with cytokines such as Eotaxin, granulocyte-colony stimulating factor (G-CSF), interleukin 7 (IL-7), migration inhibitory factor (MIF), and macrophage inflammatory protein-1α (MIP-1α). Upon single-cell RNA (scRNA) sequencing, we explored the dynamics of the adaptive immune response in few representative asymptomatic close contacts and COVID-19-infected patients. We reported direct asymptomatic contacts to have decreased CD4⁺ naive T cells with concomitant increase in CD4⁺ memory and CD8⁺ Temra cells along with expanded clonotypes compared to infected patients. Noticeable proportions of class switched memory B cells were also observed in them. Overall, these findings gave an insight into the nature of protection in asymptomatic contacts.

Keywords: SARS-CoV-2; antibody titer; cytokine levels; scBCR-seq; scRNA-seq; scTCR-seq.

Figure 1

Study approach and distinct antibody responses to SARS-CoV-2 along with neutralization assay. (A) Outline delineating the experimental workflow. (B) Box plots showing the anti-SARS-CoV-2 total, IgA, IgM, and IgG antibody proportions in serum samples from controls, symptomatic- and asymptomatic-infected individuals and in contacts (C). Pie chart showing the overall immunoglobulin response across subject groups. (D) Box plot depicting the percent inhibition of neutralizing antibodies in an in vitro spike RBD-ACE2 interaction-based surrogate virus neutralization assay. Statistical comparisons were performed using unpaired Wilcoxon test. p-values are written against respective comparisons, where n = number of individuals; IgA, immunoglobulin A; IgM, immunoglobulin M; IgG, immunoglobulin G; control (n = 14), infected (n = 23), symptomatic (n = 10), asymptomatic (n = 13), and contact (n = 24).

Figure 2

Unsupervised hierarchical clustering of antibodies and cytokines and cytokine levels with contrast of differentiation across subject groups. (A) Heatmap depicting the grouping (row-wise clustering) of individuals based on the abundance of 21 cytokines in serum and the respective antibody levels. The column-wise clustering portrays the overall coexpression of cytokines. Both row-wise clustering and column-wise hierarchical clustering were performed using Ward D method with 500 bootstraps. (B) Box plots depicting the serum cytokine levels in pg/ml (Eotaxin, G-CSF, IL-7, MIF, and MIP-1α) determined by Bio-Plex along with the (C) the area under the receiver-operating characteristic (ROC) curve of the cytokines having more than 0.8 AUC, taking 14 controls, 10 symptomatic, and 13 asymptomatic patients along with 24 contacts. Statistical comparisons were performed using unpaired Wilcoxon test; p-values are written against respective comparisons.

Figure 3

scRNA-seq analysis and demarcation of cell types according to characteristic markers. (A) UMAP representation of 7,848 single cells, color coded according to cell type. (B) Bar plot showing the percentage of cells contributed by control, contact, and infected samples. (C) Heat map showing canonical markers for individual T, B, NK, NKT, monocytes, and platelets, respectively. (D) UMAP representation of canonical markers for T cells (CD3E), B cells (MS4A1), NK (NCAM1, FCGR3A), NKT (CD3E, NCAM1), monocytes (CD14, FCGR3A), and platelets (PPBP). (E) UMAP representation of clusters stratified per individual samples: controls, contact, and patients color coded according to cell types. (F) Bar plot showing relative contribution of each of the cell types in controls, contact, and patients.

Figure 4

Phenotypical characterization of T, NK, and NKT cells. (A) UMAP representation of 5,130 single cells, color coded according to T-cell subtypes. (B) UMAP representation of subtype-specific marker genes: CD4, CCR7, FOXP3, C8A, NCAM1, GZMK, and GNLY. (C) Heatmap showing expression of marker genes for CD4⁺ naive (CCR7⁻, LEF1, TCF7, SELL), CD4 memory (AQP3, CD69), Treg (FOXP3, IL2RA), CD8 naive (CCR7, LEF1, TCF7, SELL), CD8⁺ effector GNLY (GNLY, GZMH, NKG7), effector GZMK (GZMK), CD8⁺ Temra (PTPRC, CX3CR1), gamma–delta (TRDV2, TRDC, TRGV9), NKT (NCAM1, KLRB1), and NK (NCAM1⁻ FCGR3A⁺ ). (D) Bar plot showing relative contribution of the three subclusters of CD4⁺ T cells (left), and four subclusters of CD8⁺ T cells in controls, contact, and patients (right). (E) Volcano plot showing the differentially expressed genes of CD4⁺ memory T cells between the contacts and infected patients. p-values were calculated using non-parametric Wilcoxon rank-sum test, and adjusted p-value were calculated based on the Bonferroni correction procedure. (F) GO BP enrichment analysis of differentially expressed genes upregulated in CD4⁺ memory T cells in contacts. p-values were calculated using non-parametric Wilcoxon rank-sum test, and adjusted p-value were calculated based on the Bonferroni correction procedure. (G) Volcano plot showing the differentially expressed genes of CD8⁺ Temra T cells between the contacts and infected patients. (H) GO BP enrichment analysis of differentially expressed genes upregulated in CD8⁺ Temra T cells in contacts. p-values were calculated using non-parametric Wilcoxon rank-sum test, and adjusted p-values were calculated based on the Bonferroni correction procedure.

Figure 5

Clonal expansion of T-cell receptors. (A) UMAP representation of T/NK/NKT cells colored by TCR detection. (B) Bar plot showing percentage of TCR detected in each cell cluster. (C) UMAP representation of T/NK/NKT cells colored by their clonal expansion status. Clones were defined as single (n = 1), small (1 < n ≤ 5), medium (5 < n ≤ 20), large (20 < n ≤100), and hyperexpanded (100 < n ≤ 500). (D) Nested pie plots showing the clonal status across controls, contacts, and infected patients. (E) Bar plot showing percentage of expanded clonotypes in each cluster across samples. (F) Bar plots showing usage of some TRA genes (left) and TRB genes (right) in CD8⁺ Temra cell-type across subject groups. (G) Alluvial plots showing relationship of the top V(D)J pairing frequencies of expanded clonotypes for contacts (left) and infected patient (right) between samples and cell clusters.

Figure 6

Characterization of B cells and B-cell repertoire. (A) UMAP representation of 1,626 single cells, color coded according to B-cell subtypes. (B) Heatmap showing expression of marker genes for naive B (CD19⁺ IGD⁺ CD27⁻ ), class switched memory B (CD19⁺ IGD⁻ CD27⁺ ), unswitched memory B (CD19⁺ IGD⁺ CD27⁺ ), and plasma (CD38, XBP1, MZB1). (C) Bar plot showing relative contribution of B-cell subtypes in control, contact, and infected patient samples. (D) Volcano plot showing the differentially expressed genes of class switched memory B cells between the contacts and infected patients. p-values were calculated using non-parametric Wilcoxon rank-sum test, and adjusted p-value were calculated based on the Bonferroni correction procedure. (E) GO BP enrichment analysis of differentially expressed genes upregulated in class switched memory B cells in contacts. (F) UMAP representation of B cells colored by BCR detection. (G) UMAP representation of B cells colored by their clonal expansion status. Clones were defined as single (n = 1), small (1 < n ≤ 5). (H) Nested pie plots showing the clonal status across samples. (I) Bar plot showing percentage of expanded clonotypes in each cluster in subject groups. (J) Bar plot showing percentage of IGHA, IGHM, IGHG, and IGHD isotypes in control, contact, and infected patient samples. (K) Bar plot showing usage of some IGH genes (top), IGK genes (middle), and IGL (bottom) genes (L). Heatmap showing IGH/K/L rearrangement in contact. Colors indicate the frequency of specific gene pairs.