Distinct Features and Functions of Systemic and Mucosal Humoral Immunity Among SARS-CoV-2 Convalescent Individuals

Abstract

Understanding humoral immune responses to SARS-CoV-2 infection will play a critical role in the development of vaccines and antibody-based interventions. We report systemic and mucosal antibody responses in convalescent individuals who experienced varying severity of disease. Whereas assessment of neutralization and antibody-mediated effector functions revealed polyfunctional antibody responses in serum, only robust neutralization and phagocytosis were apparent in nasal wash samples. Serum neutralization and effector functions correlated with systemic SARS-CoV-2-specific IgG response magnitude, while mucosal neutralization was associated with nasal SARS-CoV-2-specific IgA. Antibody depletion experiments support the mechanistic relevance of these correlations. Associations between nasal IgA responses, virus neutralization at the mucosa, and less severe disease suggest the importance of assessing mucosal immunity in larger natural infection cohorts. Further characterization of antibody responses at the portal of entry may define their ability to contribute to protection from infection or reduced risk of hospitalization, informing public health assessment strategies and vaccine development efforts.

Keywords: COVID-19; IgA; IgG; SARS-CoV-2; antibody; mucosal immunity; neutralization; systems serology.

Figure 1

Systemic and mucosal Ab responses. (A) Fc array characterization of IgA (left) and IgG (right) responses against a panel of SARS-CoV-2, other CoV, and control antigens in serum (top), nasal wash (middle), and stool (bottom) from convalescent (colored) and naïve (black) donors. (B) Isotypes, subclasses, and FcR binding of Abs to stabilized SARS CoV-2 S (S-2P) in serum (top) and nasal wash (bottom).

Figure 2

Mucosal and systemic Ab functions. (A, B) Functional activity of serum (A, B) nasal wash subject samples in a panel of neutralization and effector function assays including antibody-dependent phagocytosis by monocytes (ADCP) and neutrophils (ADNP), action of FcγRIIIA (ADCC), and complement cascade C3b deposition (ADCD). (C) Scatterplots of serum versus nasal wash sample activities observed for each subject for viral neutralization and RBD-specific Ab effector functions. Titer is plotted for neutralization data and peak activity is plotted for effector functions. Infection status and disease severity is indicated in color. Limit of detection (neutralization) or values observed for no Ab controls (ADCP, ADNP, ADCC) are indicated with dotted lines. No Ab controls for ADCD are indicated with the hollow black circle.

Figure 3

Correlative relationships between RBD-specific Ab features and functions. (A) Correlations observed between RBD-specific Ab features and functions in serum (left) and nasal wash (right) samples. (B) Correlations observed between Ab functions observed in serum (top) and nasal wash (bottom). (C) Representative scatterplots between highly correlated Ab features and functions in nasal wash samples. p values from unpaired two tailed t-tests with Welch’s correction. Pearson correlation coefficients (R_P) are shown.

Figure 4

Relationships among subjects and Ab features in serum and nasal wash. (A) Heatmap of filtered and hierarchically-clustered Fc array features in serum (left) and nasal wash (right) across subjects with varying infection or disease status. Each row represents an individual donor. Disease severity is shown on the left annotation bar. Each column represents an Fc Array measurement, with antigen specificity (Fv) and Fc characteristics (Fc) are indicated in top color bars. Responses are centered and scaled per feature and the scale range truncated at +/-3 SD. Relatively high responses are indicated in red, and low responses in blue. Color bars at bottom indicate the divergent clusters of IgG-related (blue) and IgA-related (purple) responses observed in nasal wash samples. (B) Representative scatter plots of the correlative relationships between IgA and IgG anti-S1 responses in nasal and serum samples. Pearson correlation coefficients (R_P) and p values of unpaired two-tailed t-tests with Welch’s correction are reported.

Figure 5

Nasal Ab features associated with disease severity. (A) Heatmap of nasal CoV-2-specific Ab features that exhibited statistically significant differences in responses between subjects with severe and non-severe (mild or moderate) disease (unpaired two-sided t-test with Welch’s correction, p < 0.05). (B, C)Representative boxplots of nasal features by disease severity across donors who experienced mild, moderate, and severe disease. (B) IgG1 specific to S-2P as a representative Ab feature elevated among subjects with severe disease. (C) FcαR binding Abs specific to RBD are highest among individuals who recovered from moderate disease as opposed to either mild or severe disease.

Figure 6

Impact of isotype depletion on antibody functions in nasal wash samples. (A–C) Scatterplots depicting the relationship between the extent of depletion of IgA (A), IgG (B), and IgM (C) and neutralization (left) and monocyte phagocytosis (right, ADCP) in nasal wash samples. Samples from four different individuals (indicated in color) were processed to deplete each of the three isotypes. Best fit line is illustrated. Spearman correlations (R_S) and two-tailed significance values are indicated in inset.