Introduction

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused more than 300,000 confirmed infections worldwide daily in the first half of 2021. Efficacy of licensed SARS-CoV-2 vaccines range from 50 to 95% depending on vaccine type and infection variant (Kim et al, 2021; McDonald et al, 2021). Thus, while vaccination markedly decreases the chances of infection and severe disease, breakthrough symptomatic and asymptomatic infections do occur. The duration of protective immunity after vaccination is uncertain, and it is unclear whether breakthrough infections are due to immune evasion by Variants of Concern (VOCs) or vaccine failure to elicit a protective immune response in some individuals (Abu-Raddad et al, 2021; Sheikh et al, 2021). In order to predict how frequently vaccine breakthrough infections could occur, and how public health outcomes would change depending on waning vaccine-mediated immunity levels, there is an urgent need to understand the immune parameters that are correlates of risk for SARS-CoV-2 vaccine breakthrough.

Recent analyses of neutralizing and binding antibody responses from cohort studies (Bergwerk et al, 2021; Feng et al, 2021; preprint: Gilbert et al, 2021) and aggregated clinical trial data (Earle et al, 2021; Khoury et al, 2021) have found correlations between antibody levels and protection from symptomatic infection. However, other potentially relevant immune parameters have not yet been investigated. Multi-parameter investigations into the levels of other important immune factors, including memory B cells and T cells, are necessary in order to better define the immune parameters that correlate with risk of vaccine breakthrough infection. In addition, correlates of risk may be different in the context of the Delta (B.1.617.2) variant which has now become the dominant strain globally (Campbell et al, 2021; World Health Organization, 2021), and shows escape from vaccine-elicited neutralizing antibody responses (Planas et al, 2021). Such correlates of risk may also be mediators of protection (Plotkin, 2010), which can be further investigated to better understand the mechanisms of vaccine-induced protection, informative for future vaccine development.

Due to extensive contact tracing efforts in Singapore, there was an opportunity to identify fully vaccinated patients who developed vaccine breakthrough infections and displayed mild symptoms or were asymptomatic. The aim of this study was to characterize the immune parameters present in this cohort of patients with mostly Delta (B.1.617.2) vaccine breakthrough infections, comparing them against those of vaccinated but uninfected close contacts in order to discern potential differences that may be correlates of risk for vaccine breakthrough.

Results

Plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 55 vaccine breakthrough cases (defined as individuals who became PCR positive at least 2 weeks after two vaccine doses) and 86 of their vaccinated and uninfected close contacts in Singapore that occurred between April and June 2021 (Table 1). Sample collection was done as soon as possible after diagnosis (maximum 7 days, and median 3 days from onset of symptoms to sample collection) (Table 1). The majority of cases (87.3%, 48/55) were identified as Delta (B.1.617.2) variant vaccine breakthrough infection via epidemiological data and direct sequencing. All participants received the Pfizer-BioNTech BNT162b2 vaccine.

Table 1. Demographics of participants. Vaccine breakthrough Close contacts Primary infection n = 55 n = 86 n = 49 Female sex 34.5% (19) 87.2% (75) 32.6% (16) Age, median (IQR) 46 (36.5–59.5) 31 (26.25–35.75) 32 (26–45) Any comorbiditya 10.9% (6) 9.3% (8) 37.0% (17) Days from symptom onset or exposure to sample collection, median (IQR)b 3 (2–4.5) 9 (7–12) 3 (2–4) Days from second dose of BNT162b2 to symptom onset or exposure, median (IQR)c 82 (51.5–99) 68 (64–70) NA Ethnicity Chinese 45.5% (25) 45.3% (39) 38.8% (19) Malay 9.1% (5) 8.1% (7) 14.3% (7) Indian 29.1% (16) 14.0% (12) 20.4% (10) Others 16.4% (9) 32.6% (28) 34.7% (17) Breakthrough variant Delta 87.3% (48) NA 0% (0) Non-Delta 5.5% (3) NA 100% (49) Unknown 7.3% (4) NA 0% (0) Severity of disease Asymptomatic 21.8% (12) NA 14.3% (7) Mild symptoms 78.2% (32) NA 85.7% (42) Severe symptoms 0% (0) NA 0% (0) Plasma antibody responses do not differ between vaccine breakthrough cases and vaccinated close contacts

We first investigated the levels of plasma antibodies against the Spike (S) protein. 100% (55/55) of vaccine breakthrough cases and 100% (86/86) of their close contacts were positive for both the Roche Elecsys® Anti-SARS-CoV-2 S and Siemens Atellica® IM SARS-CoV-2 Total (COV2T) commercial serological assays (Fig EV1A and B). These data suggest that pre-existing antibody responses were present and that these were true vaccine breakthrough cases and not cases of vaccine failure. Conversely, only 3.6% (2/55) of vaccine breakthrough cases and 1.2% (1/86) of close contacts were positive for the Roche Elecsys® Anti-SARS-CoV-2 nucleocapsid (N) commercial serological assay (Fig EV1C).

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

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. 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. 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.

Several Variants of Concern (VOCs) have emerged in the course of the COVID-19 pandemic, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). Several studies have shown reduced vaccine efficacy in populations where the predominant infection strain is a VOC (Abu-Raddad et al, 2021; Kustin et al, 2021; Lopez Bernal et al, 2021; preprint: Nasreen et al, 2021; Skowronski et al, 2022). A prior study has suggested that in the context of Alpha strain infection, lower neutralizing antibody titers correlated with the occurrence of breakthrough infections (Bergwerk et al, 2021). To determine whether neutralizing antibody titers also correlated with vaccine breakthrough risk in our cohort, we examined neutralizing antibody levels against both wild-type (WT) and Delta strains. We measured neutralizing plasma antibody responses via the surrogate virus neutralization test (sVNT), which measures inhibition of the binding between recombinant RBD protein and angiotensin-converting enzyme 2 (ACE2) (Wacharapluesadee et al, 2021). Interestingly, vaccine breakthrough cases did not display lower levels of neutralizing antibodies compared with close contacts for both the WT and Delta strains (Fig 1A). To further verify this, neutralizing plasma antibody responses were quantified via the SARS-CoV-2 pseudovirus neutralization assay and also the total IgG binding responses against membrane-anchored S-antigen via the S protein flow cytometry-based assay (SFB) (Goh et al, 2021a, 2021b). In both assays, plasma antibodies in vaccine breakthrough cases were not higher than close contacts (Fig 1B and C). Together, these data suggest that the vaccine breakthrough cases in our cohort did not have inferior plasma antibody responses against either the vaccine or breakthrough strains.

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

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. 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. 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.

Vaccine breakthrough cases have lower frequencies of RBD-specific memory B cells than vaccinated close contacts

In addition to circulating plasma antibody, memory B cells can play an important role in long-lasting immunity, including against heterologous influenza infection (Leach et al, 2019) and bacterial pneumonia (Barker et al, 2021). Thus, the frequencies of circulating SARS-CoV-2-specific memory B cells were investigated via memory B cell ELISpot targeted to the SARS-CoV-2 receptor-binding domain (RBD). In contrast to plasma antibody levels, the frequency of RBD-specific memory B cells was lower in vaccine breakthrough cases than in uninfected close contacts (Fig 2A). This was unlikely to be due to demographic differences between cohorts, since the frequencies of RBD-specific memory B cells did not correlate with age, gender, or ethnicity in either the vaccine breakthrough cases or their close contacts (Appendix Fig S1). Thus, lower circulating memory B cell levels may be a correlate of risk for vaccine breakthrough.

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

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. 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.

We further characterized the potential neutralization efficiency of antibodies secreted by memory B cells, by analyzing the levels of secreted antibody after 5 days of ex vivo stimulation with IL-2 and R848 (TLR7/8 ligand). Memory B cell supernatants containing secreted antibody were analyzed by multiplex sVNT to examine RBD-targeted neutralizing responses. Interestingly, compared to their close contacts, vaccine breakthrough cases showed higher levels of memory B cell-secreted neutralizing antibodies against both WT and Delta strains (Appendix Fig S2A). Similar observations were also found for S-specific binding antibodies measured by both the Roche S and SFB assays (Appendix Fig S2B and C). This increase may be due to memory B cell priming by ongoing infection in the vaccine breakthrough cases, which would indicate that vaccine-elicited memory B cells can be efficiently reactivated by Delta variant infection.

Memory B cells typically represent a more diverse antigen-specific population relative to plasmablasts and long-lived plasma cells (Akkaya et al, 2020). Thus, the degree of cross-reactivity in memory B cell responses relative to plasma responses was assessed. To measure the level of neutralization cross-reactivity across variants, the relative reduction in neutralization efficiency was calculated by subtracting the sVNT response against WT from the sVNT response against Delta. Compared to plasma, memory B cell-secreted antibodies retained more efficient neutralization against Delta (Fig 2B and C). This was observed in both vaccine breakthrough and vaccinated close contacts, suggesting that it is a feature of memory B cells derived from BNT162b2 vaccination. A similar trend was seen when comparing SFB plasma and memory B cell responses against each strain (Appendix Fig S2D). Together, these data suggest that antibodies from activated memory B cells are more frequently capable of cross-neutralization against Delta, as compared with plasma antibody.

To better understand the reactivation of B cell responses during breakthrough infection, we also investigated antibody-secreting plasmablast responses in a subset of the vaccine breakthrough cases and close contacts. SARS-CoV-2 RBD-specific plasmablasts were significantly higher in vaccine breakthrough cases than close contacts, as measured by antigen-specific IgG ELISpot (Fig EV2A). However, inverse correlation between memory B cells and plasmablast levels was not observed, suggesting that the reduced levels of memory B cells in vaccine breakthrough cases were not because they had differentiated into plasmablasts during ongoing infection (Fig EV2B).

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

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. 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.

Differences in CD4+ and CD8+ T cell subsets between vaccine breakthrough cases compared to vaccinated close contacts

T cell-mediated immunity is another important arm of antiviral defense (Chen & Kolls, 2013). We sought to determine whether CD4+ and CD8+ T cell profiles were distinct between vaccine breakthrough cases and their close contacts. To investigate the frequencies of virus-specific effector and memory T cells, PBMCs were stimulated in vitro using a library of SARS-CoV-2-derived peptide antigens, followed by immune phenotyping using a high-dimensional flow cytometry panel. Intracellular staining for cytokines and effector molecules (CD4+: IFNγ, IL-2, TNF; CD8+: granzyme B) was performed (Appendix Table S1, Appendix Fig S3) (Seder et al, 2008). Results showed that CD4+ T cells (CD45+ CD4+ Vδ1− Vδ2−) of vaccine breakthrough cases and close contacts were similar in their expression of IFNγ, and TNF, but vaccine breakthrough cases showed higher frequencies of CD4+ IL-2+ cells than close contacts (Fig 3A–C). Among CD8+ T cells, frequencies of granzyme B+ cells were similar between vaccine breakthrough cases and close contacts (Fig 3D). Since a polyfunctional response has been associated with effective T cell responses, we examined the frequencies of CD4+ IFNγ+ T cells that were also IL-2+ and/or TNF+ (Fig 3E). Vaccine breakthrough cases had higher frequencies of double- and triple-positive CD4+ T cells than close contacts, largely driven by the increased frequency of CD4+ IL2+ T cells in vaccine breakthrough cases. We next examined the differentiation status of peptide-stimulated T cells based on CD27 and CD45RA expression (Figs 3F and EV3). Vaccine breakthrough cases and close contacts showed similar frequencies of naïve, T central memory, T effector memory, and TEMRA cells in both CD4+ and CD8+ compartments, though there was a trend toward higher T central memory cells in vaccine breakthrough cases. Thus, vaccine breakthrough cases were broadly similar in T cell profile to close contacts, with only subtle differences observed.

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

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. 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. 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.

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Figure EV3. Differentiation status of T cells by individual

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.

Cytokine responses in vaccine breakthrough cases demonstrate non-inflammatory profile

To determine whether there were differences in inflammatory markers in vaccine breakthrough infection relative to primary infection, the levels of 45 different cytokines were also measured in the plasma of vaccine breakthrough cases and their close contacts, compared to unvaccinated patients with primary infection of matched disease severity from our earlier cohort (Table 1) (Young et al, 2020). Vaccine breakthrough cases had systemic cytokine profiles similar to their uninfected close contacts, but distinct from the primary infection (Fig 4A). Notably, cytokines commonly associated with more severe disease, including IL-1β, TNF, and IFNγ, were significantly lower compared with a previous cohort of unvaccinated patients with primary infection with matched disease symptoms (either mild symptoms or no symptoms) (Table 1; Figs 4B and EV4). Chemokines including Eotaxin, SCF, SDF-1α, and PIGF-1, associated with immune cell migration, were also significantly lower in vaccine breakthrough cases than in patients with primary infection (Figs 4B and EV4). Interestingly, healthy unvaccinated controls clustered away from both primary infection patients and vaccine breakthrough/vaccinated close contacts, suggesting different effects of infection and vaccination on innate immunity. Levels of IL-1RA were negatively correlated with memory B cell responses in vaccine breakthrough cases (Spearman correlation r = −0.674, P = 0.01) but not in close contacts (r = −0.156) (Fig EV5).

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

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. 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.

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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.

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

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.

Discussion

Here, we examine vaccine breakthrough in-depth, characterizing multiple humoral and cellular immune parameters in parallel. Differences in virus-specific plasma antibody levels between vaccine breakthrough cases and their close contacts were not observed, despite using several independent assays to examine neutralization and binding to spike protein or RBD. These are in agreement with other small studies of vaccine breakthrough cases, which found no differences or marginal differences at the plasma antibody level (Hacisuleyman et al, 2021; Mlcochova et al, 2021; Rovida et al, 2021). However, this is in contrast to recent studies from Israel (Bergwerk et al, 2021) and the UK (Feng et al, 2021) showing lower neutralizing antibody titers in patients with breakthrough Alpha variant infection and also meta-analyses showing a strong correlation between plasma antibody levels and vaccine efficacy against symptomatic infection across different vaccine types and populations (Earle et al, 2021; Khoury et al, 2021). Several reasons for this are plausible. First, the majority of cases in our study had Delta variant infection. The Delta variant is known for a greater degree of escape from vaccine-elicited neutralizing antibody responses (Planas et al, 2021), thus vaccine-elicited plasma antibodies may be less efficient for protection with different correlates of risk. Second, our study included several asymptomatic cases. Notably, in the above-mentioned vaccine efficacy study from the UK (Feng et al, 2021), no correlation was found between plasma antibody responses and asymptomatic infection, unlike with symptomatic infection (Feng et al, 2021)—different immune mechanisms may be involved to prevent asymptomatic infection.

The lower frequency of virus-specific memory B cells identified in vaccine breakthrough patients suggests that memory B cell levels against SARS-CoV-2 could be a correlate of risk for vaccine breakthrough. Previous studies have suggested that the additional diversity found in memory B cells provides a secondary layer of defense against variant pathogens that are able to escape plasma antibodies (Purtha et al, 2011; Leach et al, 2019; Akkaya et al, 2020). In line with this, we found that antibodies from activated memory B cells retained a higher fraction of their neutralizing and binding efficiency against the Delta variant, as compared to plasma antibodies. This is concordant with other recent results showing that 30-50% of monoclonal antibodies derived from memory B cells effectively bind or neutralize VOCs after BNT162b2 vaccination (Sokal et al, 2021). This suggests a potential mechanism linking memory B cells to protection against VOCs that are neutralized less efficiently by the plasma antibody response. Low frequency and/or diversity of the specific memory B cell population during exposure to SARS-CoV-2 may lead to vaccine breakthrough.

BNT162b2 vaccination elicits T cell responses (Guerrera et al, 2021), and such T cells may contribute to protection. Notably, early induction of functional anti-SARS-CoV-2 T cells after infection has been associated with rapid viral clearance and mild disease (Tan et al, 2021), raising the hypothesis that a robust T cell response could prevent virus establishment and infection. In our study, T cell responses appeared broadly similar across vaccine breakthrough and close contacts. Even though polyfunctional T cells are associated with better disease control in various infection settings (Seder et al, 2008), we did not identify a lack of polyfunctionality in the CD4+ T cells of vaccine breakthrough cases.

Vaccine breakthrough cases showed cytokine profiles that were remarkably similar to the uninfected vaccinated close contact cohort and dissimilar to patients who contracted primary infection with mild symptoms. This provides further evidence for immune-mediated protection against virus-induced inflammation and disease despite vaccine breakthrough infection. Notably, memory B cell responses were negatively correlated with IL-1RA levels in vaccine breakthrough cases but not in close contacts. IL-1RA has been previously associated with disease severity (Zhao et al, 2020), suggesting that memory B cell responses may play a role in alleviating inflammation and disease severity despite vaccine breakthrough.

The primary limitation of the current study is in its retrospective design and lack of pre-infection immune status. Such studies will not be able to rule out confounding effects due to early events in the response to infection. These include boosts to antibody levels due to activation of antibody-secreting cells, as well as immediate changes to circulating memory B cell levels and T cell levels due to recruitment into germinal centers or other lymphoid organs. In particular, reduced memory B cell levels in vaccine breakthrough cases may have been due to recruitment of memory B cells out of circulation after activation by infection. The current study provides additional impetus for future prospective studies to closely examine memory B cell and T cell responses. Studies have indicated that memory B cell responses are longer lasting compared with plasma antibody levels following natural infection (Dan et al, 2021; Sakharkar et al, 2021; Turner et al, 2021). Memory B cell responses are also effectively elicited by vaccination (Goel et al, 2021; Piano Mortari et al, 2021), though their longevity relative to plasma antibody remains an open question. Protective effects of memory B cells may become more apparent with increased time since vaccination, as plasma antibody levels wane. Should vaccine-elicited memory B cell responses prove to be long-lived, and also a mechanistic correlate of protection, vaccines may provide a longer duration of protection compared to what might be expected based on plasma antibody levels alone.

Materials and Methods Ethics statement

The study design and protocols for the COVID-19 PROTECT study group were evaluated by National Healthcare Group (NHG) Domain Specific Review Board (DSRB) and approved under study number 2012/00917. Collection of healthy donor samples was approved by SingHealth Centralized Institutional Review Board (CIRB) under study number 2017/2806 and NUS IRB 04-140. Written informed consent was obtained from participants in accordance with the Declaration of Helsinki for Human Research. The experiments conformed to the principles set out in the Department of Health and Human Services Belmont Report.

Clinical data and biological sample collection

A total of 55 vaccine breakthrough cases (infected at least 14 days after two doses of vaccine) and 86 of their close contacts (exposed at least 14 days after two doses of vaccine) were recruited into the study from April to June 2021. All vaccine breakthrough cases and close contacts were vaccinated with the Pfizer BNT162b2 vaccine. Demographic data, disease severity, and clinical laboratory data were obtained from patient records throughout hospitalization or during quarantine (Table 1). Blood was collected in Cell Preparation Tubes (CPT) (Becton Dickinson) as soon as possible after infection was confirmed by PCR. Blood samples were also collected from their uninfected close contacts during early time of quarantine. The plasma fraction was extracted for antibody and cytokine analyses, and isolated peripheral blood mononuclear cells (PBMCs) were used for flow cytometry staining for immune cell phenotyping, as well as B cell ELISpot for memory B cell and plasmablast quantification. Antibody and cellular assays were performed subject to sample availability. Plasma samples from a total of 49 patients with primary infection, who were recruited into our earlier cohort from February to August 2020, were also included in our cytokine analysis.

Serological measurement of SARS-CoV-2 antibodies

Plasma specimens (n = 55 vaccine breakthrough, n = 86 close contact) were stored at −25°C and equilibrated at room temperature before time of analysis. Specimens were analyzed in accordance with the manufacturer’s protocol.

The Elecsys® Anti-SARS-CoV-2 S (Roche S) and Elecsys® Anti-SARS-CoV-2 (Roche N) immunoassays were implemented on the Roche cobas e411 Analyzer (Roche) for the quantitative detection of total antibodies against the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) and the qualitative detection of total antibodies against the SARS-CoV-2 nucleocapsid (N) antigen, respectively. Serum samples were incubated with either a mix of biotinylated and ruthenylated SARS-CoV-2 S-RBD antigens or N antigens, corresponding to the test required, to form immune complexes. The complexes w