Decreased memory B cell frequencies in COVID-19 delta variant vaccine breakthrough infection

Abstract

The SARS-CoV-2 Delta (B.1.617.2) variant is capable of infecting vaccinated persons. An open question remains as to whether deficiencies in specific vaccine-elicited immune responses result in susceptibility to vaccine breakthrough infection. We investigated 55 vaccine breakthrough infection cases (mostly Delta) in Singapore, comparing them against 86 vaccinated close contacts who did not contract infection. Vaccine breakthrough cases showed lower memory B cell frequencies against SARS-CoV-2 receptor-binding domain (RBD). Compared to plasma antibodies, antibodies secreted by memory B cells retained a higher fraction of neutralizing properties against the Delta variant. Inflammatory cytokines including IL-1β and TNF were lower in vaccine breakthrough infections than primary infection of similar disease severity, underscoring the usefulness of vaccination in preventing inflammation. This report highlights the importance of memory B cells against vaccine breakthrough and suggests that lower memory B cell levels may be a correlate of risk for Delta vaccine breakthrough infection.

Keywords: COVID-19; correlate of risk; delta; memory B cells; vaccine breakthrough.

Figure EV1. Anti‐SARS‐CoV‐2 titers in commercial serological assays

1. Anti‐SARS‐CoV‐2 S protein antibodies in plasma of vaccine breakthrough cases (n = 55) or close contacts (n = 86) were determined by Roche Elecsys® S antibody assay. Values above the upper limit of quantitation, 250 U/ml, are truncated to 250 U/ml.

2. Anti‐SARS‐CoV‐2 S1 RBD antibodies in plasma of vaccine breakthrough cases (n = 55) or close contacts (n = 86) determined by Siemens Atellica® IM SARS‐CoV‐2 Total (COVT) assay. Values above the upper limit of quantitation, 10, are truncated to 10.

3. Anti‐SARS‐CoV‐2 N protein antibodies in plasma of vaccine breakthrough cases (n = 55) or close contacts (n = 86) determined by Roche Elecsys® N antibody assay.

Data information: Dotted lines indicate upper limit of quantitation (A,B) or positivity threshold (B,C) based on manufacturer’s instructions. Error bars indicate median and interquartile range. Source data are available online for this figure.

Figure 1. Similar plasma antibody levels against SARS‐Cov‐2 between vaccine breakthrough cases and close contacts

1. Anti‐SARS‐CoV‐2 neutralizing antibodies in plasma of vaccine breakthrough cases (n = 54) and close contacts (n = 86) were measured by surrogate virus neutralization test (sVNT). The percentage inhibition of ACE2 binding to RBD at plasma dilution of 1:800 is given. The wild‐type (Wuhan) strain or Delta (B.1.617.2) strain RBD was used, respectively.

2. Anti‐SARS‐CoV‐2 neutralizing antibodies in plasma of vaccine breakthrough cases (n = 29) and close contacts (n = 86) were measured by pseudovirus neutralization assay.

3. Anti‐SARS‐CoV‐2 S protein antibodies in plasma of vaccine breakthrough cases (n = 48) and close contacts (n = 86) were determined by S protein flow cytometry‐based assay (SFB). The percentage of plasma antibody‐bound cells out of the total population of cells transfected with membrane‐anchored SARS‐CoV‐2 S protein is given. In all graphs, error bars denote median and interquartile range. Red solid circles indicate Delta strain vaccine breakthrough, and red open circles indicate non‐Delta or unknown strain.

Data information: Statistical comparisons were determined by one‐tailed Mann–Whitney U‐test. Error bars indicate median and interquartile range. Source data are available online for this figure.

Figure 2. Memory B cell responses against SARS‐CoV‐2 in vaccine breakthrough cases and close contacts

1. A

   Frequencies of memory B cells specific for SARS‐CoV‐2 RBD are examined via ELISpot for vaccine breakthrough cases (n = 25) and close contacts (n = 86). The frequency of RBD‐specific memory B cells is given as a percentage of the total IgG‐secreting B cell population. P value was determined by two‐tailed Mann–Whitney U‐test, **P < 0.01. Error bars indicate median and interquartile range.

2. B, C

   Levels of anti‐SARS‐CoV‐2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate virus neutralization test (sVNT), in supernatant from 5‐day culture of activated memory B cells and in plasma. The difference in sVNT % inhibition between WT and Delta is taken, and this difference is compared between plasma and memory B cell supernatant for vaccine breakthrough cases (n = 48 pairs) (B) and for close contacts (n = 86 pairs) (C).

Data information: P values for paired comparisons were determined by two‐tailed Wilcoxon matched‐pairs signed rank test, ****P < 0.0001. In all graphs, error bars denote median and interquartile range. Red solid circles indicate Delta strain vaccine breakthrough, and red open circles indicate non‐Delta or unknown strain. Source data are available online for this figure.

Figure EV2. SARS‐CoV‐2 RBD‐specific plasmablasts are increased in vaccine breakthrough participants

1. Frequencies of plasmablasts specific for SARS‐CoV‐2 RBD are examined via ELISpot for vaccine breakthrough cases (n = 24) and close contacts (n = 86). The frequency of RBD‐specific memory B cells is given as the number of plasmablasts (as determined by spot‐forming units, SFU) per 1,000 PBMCs plated. Error bars denote median and interquartile range. P value for unpaired comparison was determined by two‐tailed Mann–Whitney U‐test, ****P < 0.0001.

2. The relationship between SARS‐CoV‐2 RBD‐specific plasmablast and memory B cell frequencies in vaccine breakthrough cases was quantified via ELISpot is shown, and correlation was determined by Spearman correlation (n = 7 pairs). Red solid circles indicate Delta strain vaccine breakthrough, and red open circles indicate non‐Delta or unknown strain.

Source data are available online for this figure.

Figure 3. T cell responses in vaccine breakthrough cases and close contacts

1. A–D

   PBMCs from vaccine breakthrough cases (n = 15) and close contacts (n = 26) were examined for T cell responses. PBMCs were left unstimulated (Unstim), were stimulated with pooled SARS‐CoV‐2 PepTivator^® S, S1, M, and N peptides for 6 h (SARS‐CoV‐2 Peptide Stim), or non‐specifically stimulated with phorbol 12‐myristate 13‐acetate (PMA), then assessed by high‐dimensional flow cytometry. CD4⁺ T cells positive for intracellular staining of IFNγ (a), IL‐2 (b), TNF (c), or CD8⁺ T cells positive for intracellular staining of granzyme B (d) are shown. Error bars indicate median and interquartile range.

2. E

   To examine polyfunctionality of CD4⁺ T cells, SARS‐CoV‐2 peptide‐stimulated IFNγ⁺ CD4⁺ T cells were further examined for co‐expression of IL‐2 and/or TNF, and the fraction of cells expressing IFNγ only (1 function), IFNγ and either IL‐2 or TNF (2 functions), or IFNγ and both IL‐2 and TNF (3 functions) are shown.

3. F

   The differentiation status of SARS‐CoV‐2 peptide‐stimulated CD4⁺ T cells (left) and CD8⁺ T cells (right) were compared based on CD27 and CD45RA expression (Naïve: CD27⁺ CD45RA⁺; T central memory (TCM): CD27⁺ CD45RA⁻; T effector memory (TEM): CD27⁻ CD45RA⁻; TEMRA: CD27⁻ CD45RA⁺). The average of all vaccine breakthrough cases or close contacts is plotted, respectively.

Data information: P value for unpaired comparison was determined by two‐tailed Mann–Whitney U‐test, **P < 0.01. Source data are available online for this figure.

Figure EV3. Differentiation status of T cells by individual

1. A, B

   PBMCs from vaccine breakthrough cases (n = 15) and close contacts (n = 26) were examined for T cell responses. PBMCs were left unstimulated (Unstim), were stimulated with pooled SARS‐CoV‐2 PepTivator^® S, S1, M, and N peptides for 6 h (SARS‐CoV‐2 Peptide Stim), or non‐specifically stimulated with phorbol 12‐myristate 13‐acetate (PMA), then assessed by high‐dimensional flow cytometry. The differentiation status of CD4⁺ T cells (A) and CD8⁺ T cells (B) in unstimulated, SARS‐CoV‐2 peptide‐stimulated, and PMA‐stimulated conditions were compared based on CD27 and CD45RA expression (Naïve: CD27⁺ CD45RA⁺; T central memory (TCM): CD27⁺ CD45RA⁻; T effector memory (TEM): CD27⁻ CD45RA⁻; TEMRA: CD27⁻ CD45RA⁺). Error bars indicate median and interquartile range.

Source data are available online for this figure.

Figure 4. Plasma immune mediator levels of COVID‐19 vaccine breakthrough cases

1. Levels of specific immune mediators in the first plasma samples collected during hospitalization or quarantine were quantified using a 45‐plex microbead‐based immunoassay. PCA of 45 immune mediator levels analyzed in COVID‐19 vaccine breakthrough cases (n = 39) compared to their vaccinated close contacts (n = 32), unvaccinated COVID‐19 patients with primary infection (n = 49), and healthy unvaccinated controls (n = 24). PC1 explains 21.6% of the variation, while PC2 explains 9.4% of the variation; color denotes different groups of subjects.

2. Heat map of selected immune mediator levels in plasma samples of healthy controls, vaccinated close contacts, COVID‐19 vaccine breakthrough cases, and unvaccinated COVID‐19 patients with primary infection. Each color represents the relative concentration of a particular analyte. Blue and red indicate low and high concentration, respectively.

Source data are available online for this figure.

Figure EV4. Cytokine responses in vaccine breakthrough cases and close contacts show milder inflammatory profile compared to primary infection in unvaccinated persons

Cytokine levels in vaccine breakthrough cases, close contacts, and a matched primary infection cohort were determined by Luminex assay. Selected inflammation‐related (top row) and cell migration‐related (bottom row) cytokines are shown. Dotted lines represent the average response in a population of healthy controls. In all graphs, error bars denote median and interquartile range. P values for unpaired comparisons were determined by two‐tailed Mann–Whitney U‐test, *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.

Figure EV5. Correlation between IL‐1RA levels and SARS‐CoV‐2 RBD‐specific memory B cells

1. A, B

   The relationship between IL‐1RA and log‐transformed SARS‐CoV‐2 RBD‐specific memory B cell responses is shown for vaccine breakthrough patients (A) and in their close contacts (B). Dotted lines represent the baseline of 0. Correlation was determined by Spearman correlation, *P < 0.05.

Source data are available online for this figure.