Rapid generation of durable B cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence

Abstract

Lasting immunity following SARS-CoV-2 infection is questioned because serum antibodies decline in convalescence. However, functional immunity is mediated by long-lived memory T and B (Bmem) cells. Therefore, we generated fluorescently-labeled tetramers of the spike receptor binding domain (RBD) and nucleocapsid protein (NCP) to determine the longevity and immunophenotype of SARS-CoV-2-specific Bmem cells in COVID-19 patients. A total of 36 blood samples were obtained from 25 COVID-19 patients between 4 and 242 days post-symptom onset including 11 paired samples. While serum IgG to RBD and NCP was identified in all patients, antibody levels began declining at 20 days post-symptom onset. RBD- and NCP-specific Bmem cells predominantly expressed IgM⁺ or IgG1⁺ and continued to rise until 150 days. RBD-specific IgG⁺ Bmem were predominantly CD27⁺, and numbers significantly correlated with circulating follicular helper T cell numbers. Thus, the SARS-CoV-2 antibody response contracts in convalescence with persistence of RBD- and NCP-specific Bmem cells. Flow cytometric detection of SARS-CoV-2-specific Bmem cells enables detection of long-term immune memory following infection or vaccination for COVID-19.

Fig. 1. Construct design and detection of SARS-CoV-2 RBD- and NCP-specific B cells

(A) Recombinant spike receptor binding domain (RBD) and nucleocapsid protein (NCP) constructs of SARS-CoV-2. (B) SDS PAGE of purified, reduced (R) or non-reduced (NR) recombinant RBD and NCP. (C) Flow cytometry stainings of CD19⁺ B cells from an uninfected control and a recovered COVID-19 patient using double discrimination through inclusion of two fluorescent tetramers for each protein (RBD or NCP) in the same staining tube. Percentages indicate the proportions of RBD- or NCP-specific cells within total CD19⁺ B cells.

Fig. 2. Neutralizing antibodies and RBD-, and NCP- and HA-specific IgG antibody levels.

(A) Neutralizing antibody titers to SARS-CoV-2 in 25 COVID-19 patients and 36 historic controls (sampled in 2019 and Q1 2020) as determined using a pseudovirus assay. Antigen-specific plasma IgG levels were determined to (B) SARS-CoV-2 RBD, (C) SARS-CoV-2 NCP and (D) influenza A/Michigan/2015 haemagglutinin (HA). Horizontal solid gray lines represent median values. (E) Neutralizing antibody titers, and IgG levels to (F) RBD and (G) NCP plotted against time since symptom onset of infection of 25 patients including 11 patients sampled twice. Patient datapoints are marked based on disease severity with severe as red triangles, moderate as orange diamonds and mild as blue circles. The 11 paired samples are connected with black lines. The dotted horizontal lines in panels A and E depict an ID50 of 20, the cut-off for neutralization (68). The dotted horizontal lines in B, C, F and G depict the cut-off for positivity, defined as +2SD of the controls. Statistics were performed with the Mann-Whitney U-test for unpaired data; **** p ≤ 0.0001.

Fig. 3. RBD- and NCP-specific Bmem cells predominantly express IgM or IgG1.

(A) Gating strategy to discriminate T and B cells, followed by subsetting of total B cells into CD27^-IgD⁺ naive, CD27⁺IgD⁺ Bmem and CD27^+/−IgD^- Bmem cells. Within IgD^- Bmem cells, Ig switched subsets were defined based on the differential expression of IgG1, 2, 3, 4 subclasses and IgA. (B) Detection of RBD-specific (RBD⁺) B cells, and (C) NCP-specific (NCP⁺) B cells, utilized the same gating strategy as for total B cells. (D) Absolute numbers of IgG⁺ RBD⁺ and NCP⁺ Bmem cells in the first sample of 25 COVID-19 patients and 10 uninfected healthy controls. (E) Median frequencies of total, RBD⁺ and NCP⁺ Bmem subsets in 25 COVID-19 patients. Significant differences between RBD⁺ and NCP⁺ Bmem subsets are depicted with asterisks in the NCP column. (F) Frequencies of IgG⁺ Bmem cells expressing CD27 within total, RBD⁺ and NCP⁺ Bmem cells. Patient datapoints are marked based on disease severity with severe as red triangles, moderate as orange diamonds and mild as blue circles. Statistics: panel D, Mann-Whitney U-test for unpaired data; panels E and F, Wilcoxon matched-pairs signed rank test for paired samples; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 4. Composition and kinetics of SARS-CoV-2 RBD- and NCP-specific Bmem in convalescence.

Relative composition of the Bmem cell compartment based on Ig isotype and IgG subclass expression within (A) RBD-specific (RBD⁺) and (B) NCP-specific (NCP⁺) Bmem subsets. Patients’ data are ordered by days post-symptom onset. Absolute numbers of total, IgM⁺ and IgG⁺ Bmem cells specific for (C) RBD⁺ or (D) NCP⁺. Samples are plotted by days post-symptom onset for 25 individuals, with 11 patients sampled twice and paired samples connected with gray lines. Patient datapoints are marked based on disease severity with severe as red triangles, moderate as orange diamonds and mild as blue circles.