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The ongoing evolution and emergence of SARS-CoV-2 variants raise concerns about the effectiveness of monoclonal antibody therapies and vaccines. The mRNA-based vaccines Pfizer-BioNTech BNT162b2 and Moderna mRNA-1273 encode a stabilized full-length SARS-CoV-2 spike ectodomain derived from the Wuhan-Hu-1 genetic sequence and elicit potent neutralizing antibodies (NAbs)7,8. However, emerging SARS-CoV-2 variants with mutations in the spike gene, especially in NAb-binding sites, have been associated with increased transmissibility9,10 as well as neutralization resistance to monoclonal antibodies, convalescent plasma and sera from vaccinated individuals1,2,3,4,5,6. To better understand how immune responses triggered by vaccination and/or SARS-CoV-2 infection fare against emerging virus variants, we assembled a cohort of mRNA-vaccinated individuals, previously infected or not, and characterized virus-specific immunological profiles. We examined the effect of SARS-CoV-2 variants containing many different key spike gene mutations in mRNA-vaccinated individuals using a comprehensive set of full-length authentic SARS-CoV-2 isolates.

Vaccine-induced antibody responses

First, to characterize SARS-CoV-2-specific adaptive immune responses after mRNA COVID-19 vaccines (Moderna or Pfizer), 40 healthcare workers (HCWs) from the Yale-New Haven Hospital were enrolled in this study between November 2020 and January 2021, with a total of 198 samples. We stratified the vaccinated participants based on previous exposure to SARS-CoV-2 into previously infected (recovered) or uninfected (naive) groups. Previous infection was confirmed by reverse-transcription quantitative PCR (RT–qPCR) and SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA). The HCWs received mRNA vaccines, either Pfizer or Moderna, and we followed them longitudinally pre-vaccination and post-vaccination (Fig. 1a). Cohort demographics, vaccination status and serostatus are summarized in Extended Data Table 1. We collected plasma and peripheral blood mononuclear cells (PBMCs) sequentially in five time points that covered a period of 98 days after the first vaccination dose. Samples were collected at baseline (before vaccination), 7 and 28 days after the first vaccination dose, and 7, 28 and 70 days after the second vaccination dose (Fig. 1a). We determined antibody profiles, using both ELISA and neutralization assays, and assessed cellular immune response, profiled by flow cytometry using frozen PBMCs.

Fig. 1: Temporal dynamics of anti-SARS-CoV-2 antibodies in vaccinated participants.
figure 1

a, Cohort timeline overview indicated by days after SARS-CoV-2 mRNA vaccination. HCW participants received two doses of the mRNA vaccine and plasma samples were collected as indicated. Baseline (time point 0 (TP 0)), before vaccination; TP 1, 7 days after the first dose; TP 2, 28 days after the first dose; TP 3, 7 days after the second dose; TP 4, 28 days after the second dose; TP 5, 70 days after the second dose. Participants were stratified on the basis of previous exposure to SARS-CoV-2 (purple: vaccinated, uninfected; blue: vaccinated, previously infected). Created with BioRender.com. b, c, Plasma reactivity to the spike (S) protein, receptor-binding domain (RBD) and nucleocapsid (N) measured over time by ELISA. The levels of anti-spike, anti-spike subunit 1 (S1), anti-RBD and anti-nucleocapsid IgG are shown (b). A comparison of the levels of anti-spike, anti-S1, anti-RBD and anti-nucleocapsid IgG in vaccinated participants previously infected or not to SARS-CoV-2 is also displayed (c). d, e, Longitudinal neutralization assay using wild-type SARS-CoV-2, ancestral strain (WA1, USA). The neutralization titre (PRNT50) over time (d), and the plasma neutralization capacity between vaccinated participants previously infected or not to SARS-CoV-2 (e) are shown. In b and d, significance was assessed by one-way analysis of variance (ANOVA) corrected for multiple comparisons using Tukey’s method. The bars represent mean values ± standard deviations. In c and e, longitudinal data are plotted over time continuously. Regression lines are shown as blue (previously infected) and purple (uninfected). The lines indicate cross-sectional averages from each group with shading representing 95% CI and are coloured accordingly. Significance was assessed using unpaired two-tailed t-test. TP 0: n = 37; TP 1: n = 35; TP 2: n = 30; TP 3: n = 34; TP 4: n = 31; TP 5: n = 27). Each dot represents a single individual. ****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05.

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We found that mRNA vaccines induced high titres of virus-specific antibodies that declined over time, as previously reported6,11 (Fig. 1b, c). After the first vaccine dose, over 97% of vaccinated participants developed virus-specific IgG titres, which increased to 100% after the second dose. IgG titres against the spike protein, spike subunit 1 and receptor-binding domain (RBD) peaked 7 days after the second dose of the vaccine (Fig. 1b, c). No differences were observed in antibody levels between vaccinated participants of different sexes and after stratification by age (Extended Data Fig. 1a). Consistent with previous reports7,12, we found that virus-specific IgG levels were significantly higher in the vaccinated group who were previously infected than in the vaccinated group who were uninfected (Extended Data Fig. 1b, c). As expected, given the absence of sequences encoding nucleocapsid antigens in the mRNA vaccines, anti-nucleocapsid antibody titres remain stable over time for vaccinated individuals who were previously infected, and were not affected by vaccination in both the uninfected and the previously infected groups (Fig. 1b, c). We next assessed plasma neutralization activity longitudinally against an authentic SARS-CoV-2 strain USA-WA1/2020 (lineage A), with a similar spike gene amino acid sequence as the Wuhan-Hu-1 sequence that was used for the mRNA vaccine design, by a 50% plaque-reduction neutralization (PRNT50) assay. Neutralization activity directly correlated with anti-spike and anti-RBD antibody titres, also peaking at 7 days after the second dose of the vaccine (Fig. 1d, e). However, both groups displayed similar neutralization titres against the lineage A virus isolate at the peak of response (Fig. 1d, e). Our data indicate that despite faster and more exuberant antibody responses to viral proteins by vaccinated individuals who were previously infected than vaccinated individuals who were uninfected, vaccination led to overall similar levels of NAbs after the second dose of the vaccine.

Vaccine-induced T cell responses

A robust T cell response has also been linked to efficient protective immunity against SARS-CoV-2 (refs. 13,14,15). Hence, we next longitudinally analysed spike-reactive and nucleocapsid-reactive T cell responses in vaccinated individuals. To detect low-frequency peptide-specific T cell populations, we first expanded antigen-specific T cells by stimulating PBMCs from vaccinated individuals with spike and nucleocapsid peptide pools ex vivo for 6 days, followed by restimulation with the same peptide pools and analysis of activation markers after 12 h. To cover the entire spike protein, two peptide pools (S-I and S-II) were used, while a single peptide pool was used for nucleocapsid stimulation. Spike-reactive CD4+ and CD8+ T cells increased over time following vaccination (Fig. 2a, b), as evidenced by an increase in the number of cells expressing the activation markers CD38 and HLA-DR; no differences were observed between the previously infected and uninfected vaccinated groups. Consistent with previous reports16,17, spike-reactive CD4+ T cell responses were comparable among full-length lineage A and P.1 virus isolates. By contrast, spike-reactive CD8+ T cell responses were only observed to lineage A, and not P.1, 28 days after the second dose of the vaccine, suggesting that spike-specific CD8+ T cell responses can be affected by the mutations within the spike gene of SARS-CoV-2 variants (Fig. 2b). As expected, nucleocapsid-reactive T cells induced after stimulation with a nucleocapsid peptide pool derived from the lineage A virus isolate were primarily observed in vaccinated individuals who were previously infected (Fig. 2b). We also observed elevated numbers of nucleocapsid-reactive CD4+ T cells in previously infected individuals at 28 days after the second dose of the vaccine, paralleling general activation of CD4+ T cells (Fig. 2b, Extended Data Fig. 2a). Moreover, we observed increased counts of activated CD4+ T cell, follicular helper T cell and antibody-secreting cells 28 days after the booster vaccine (Extended Data Fig. 2). Thus, T cell responses in vaccinated individuals display similar dynamics as antibody responses.

Fig. 2: Temporal dynamics of anti-SARS-CoV-2 T cell immunity in vaccinated participants.
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SARS-CoV-2 S-reactive CD4+ and CD8+ T cells after in vitro stimulation with SARS-CoV-2 S-I and S-II peptide pools and nucleoprotein peptide pool. a, Representative dot plots from four vaccinated individuals, 28 days after the second dose of the vaccine, showing the percentage of double-positive cells expressing HLA-DR and CD38 out of CD4+ T cells (top) and CD8+ T cells (bottom). Individuals who were previously infected to SARS-CoV-2 or uninfected are indicated by blue or purple shades, respectively. b, The percentage of double-positive cells, spike-reactive and nucleocapsid-reactive out of CD4+ T cells (top) and CD8+ T cells (bottom) over time post-vaccination. Individuals who were previously infected to SARS-CoV-2 or uninfected are indicated by blue or purple dots, respectively. Each dot represents a single individual. Significance was assessed by one-way ANOVA corrected for multiple comparisons using Dunnett’s method. Vaccination time points were compared with baseline. Stimulation values were subtracted from the respective non-stimulation condition. The bars represent the distribution of variables with quartiles and outliers. The horizontal bars indicate mean values. TP 0: n = 30; TP 1: n = 34; TP 2: n = 27; TP 3: n = 27; TP 4: n = 24). Non-stim, non-stimulated PBMCs; nucleocapsid, PBMCs stimulated with the SARS-CoV-2 nucleocapsid protein pool derived from the ancestral lineage A virus, WA1, USA; spike, PBMCs stimulated with the SARS-CoV-2 spike protein pool derived from the ancestral strain lineage A, WA1, USA; spike (P.1), PBMCs stimulated with the SARS-CoV-2 spike protein pool derived from the P.1 variant. ****P < 0.0001, ***P < 0.001 and **P < 0.01.

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Vaccine-induced NAb against variants

To investigate potential differences in NAb escape between the SARS-CoV-2 variants, we analysed the neutralization capacity of plasma samples from vaccinated individuals against a panel of 18 genetically distinct and authentic SARS-CoV-2 isolates. Among the isolates, 16 were from our Connecticut SARS-CoV-2 genomic surveillance programme representing variants from the same geographical region as our HCW cohort18. Our variant panel includes representatives of all lineages currently classified as variants of concern (B.1.1.7, B.1.351, P.1 and B.1.617.2) as well as lineages classified as variants of interest (B.1.427, B.1.429, B.1.525, B.1.526 and B.1.617.1)19. In addition, we selected lineages with key spike gene mutations (B.1.517 with N501T, and B.1 and R.1 with E484K)20, and included lineage A as a comparison (Fig. 3a). To help deconvolute the effects of individual mutations, we included four different B.1.526 isolates (labelled as B.1.526a–d) that represent different phylogenetic clades and key spike gene mutations (L452R, S477N and E484K) (Extended Data Fig. 3b), two different B.1.1.7 isolates with (B.1.1.7b) and without (B.1.1.7a; most common) E484K, and two B.1.351 isolates with (B.1.351b) and without (B.1.351a; most common) L18F (Fig. 3a, Extended Data Table 2). Except lineage A, all isolates (lineages B, P and R) have the spike gene D614G mutation located in the receptor-binding motif, which has been reported to have a modest effect on vaccine-elicited neutralization21. For each isolate, we highlight key spike protein amino acid differences in the antigenic regions of the amino-terminal domain (NTD), the RBD–ACE2 interface and the furin cleavage site (Fig. 3a). A full list of amino acid substitutions and deletions from all genes is provided in Extended Data Table 2. We used a PRNT50 assay to determine the neutralization titres of plasma collected 28 days after the second dose of the vaccine.

Fig. 3: Effect of SARS-CoV-2 variants of concern on neutralization capacity of vaccinated participants.
figure 3

a, Plasma neutralization titres against ancestral lineage A virus (WA1, USA) and locally circulating variants. Sixteen SARS-CoV-2 variants were isolated from nasopharyngeal swabs of individuals who were infected and an additional B.1.351 isolate was obtained from BEI. Neutralization capacity was assessed using plasma samples from vaccinated participants, 28 days after the second dose of the SARS-CoV-2 vaccine at the experimental sixfold serial dilutions (from 1:3 to 1:2,430). Key spike mutations within the distinct lineages and plasma neutralization titres (PRNT50) are also shown. Spike mutations are arranged across columns and the rows represent lineages. Significance was assessed by one-way ANOVA corrected for multiple comparisons using Dunnett’s method. Neutralization capacity of variants was compared with the neutralization capacity against the ancestral strain. The bars represent mean values ± standard deviations, and the dotted line indicates the mean value of PRNT50 to the ancestral strain. Superscripted letters indicate different isolates belonging to the same lineage, with distinct spike amino acid changes. n = 32. FCS, furin cleavage site; RBM, receptor-binding motif. b, Estimated effect of individual mutations on plasma neutralization titres. Neutralization estimates (log PRNT50) and significance were tested with a linear mixed model with subject-level random effects. The dots represent the ratio of linear mixed model coefficients plus intercept to the intercept alone, the error bars represent the standard error, and the dotted line indicates no estimated effect on neutralization. c, Individual trajectories of plasma neutralization titres (PRNT50). Each line represents a single individual. n = 32. Variants were grouped giving specific spike mutations and are coloured accordingly. Superscripted letters indicate different isolates belonging to the same lineage, with distinct spike amino acid changes. ****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05.

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When comparing vaccine-induced neutralization against the different isolates in comparison to the lineage A virus isolate, we observed significantly reduced PRNT50 titres for 12 out of the 17 isolates, and the rank order of reduced neutralization mostly clustered by key spike gene amino acid differences (Fig. 3a). Virus isolates with both the E484K and the N501Y (or N501T) mutations (B.1.351b, B.1.351a, B.1.1.7b, B.1 and P.1) reduced neutralization the most (4.6–6.0-fold decrease in PRNT50 titres). Virus isolates with the L452R mutation (B.1.617.1, B.1.429, B.1.526b, B.1.427 and B.1.617.2) were in the next grouping of decreased neutralization (2.5–4.1-fold decrease), which partially overlapped with isolates with E484K but without N501Y/T (B.1.525, R.1 and B.1.526c; 2.0–3.8-fold decrease). To further assess the effect of individual mutations, we constructed a linear mixed model with subject-level random effects to account for the differences in neutralization outcome (log-transformed PRNT50 titres) by each individual mutation as compared with lineage A (with no mutation) (Fig. 3b). From our model, we estimated that 8 of the 11 key spike gene mutations that we investigated had significant negative effects on neutralization, and that L452R (2.8-fold decrease in PRNT50 titres; P < 2 × 10–16) and E484K/Q (2.0-fold decrease; P < 2 × 10–16) had the greatest individual effects. As combinations of mutations can alter effects differently than the added value of each individually (that is, epistatic interactions), we also created a second linear mixed model that controlled for all of the individual mutations in the first model as well as three common combinations of key spike gene mutations found in our isolates: ΔH69/V70 and E484K, L452R and P681R, and E484K and N501Y. These combinations of mutations allowed us to assess whether the contribution of mutations together is synergistic, antagonistic or neither. Our model suggests that the ΔH69/V70 and E484K combination was synergistic (that is, decreased neutralization more than the added effects of each; β = −0.182; P = 0.005), L452R and P681R was antagonistic (that is, decreased neutralization less than the added effects of each; β = 0.228; P = 0.003), and E484K and N501Y was neither (that is, neutralization was probably the sum of the individual effects of each; β = 0.060; P = 0.248). Thus, from our large panel of virus isolates, we find that virus genotype has an important role in vaccine-induced neutralization, with L452R probably having the largest individual effect, but the added effects of E484K and N501Y make viruses with this combination perhaps the most concerning for vaccines.

Although virus-specific factors may have a significant role in neutralization, differences in neutralization activity between individual vaccinated HCWs were much larger (up to approximately 2 log PRNT50 titres) than differences among virus isolates (mostly less than 1 log) (Fig. 3a). By tracking PRNT50 titres from each HCW, we found that the vaccinated individuals with high neutralization activity for the lineage A virus isolate are typically on the higher end of neutralization for all variants (Fig. 3c). Moreover, two vaccinated HCWs did not develop NAbs against any of the virus isolates, including lineage A (Fig. 3c), despite the production of virus-specific antibodies (Fig. 1a, b).

Effect of previous infection on NAbs

To further understand the underlying factors that determine the levels of neutralization activity, we separated individuals by their previous SARS-CoV-2 infection status (that is, previously infected versus uninfected) and determined their neutralization titres to our panel of SARS-CoV-2 isolates. Previous infection occurred between April and December 2020, and for 3 out of 15 HCWs, we were able to identify the lineage of the virus, which were all B.1.3. While the rank order of virus isolates affecting neutralization remained mostly the same, we found that the plasma from vaccinated individuals who were previously infected generally had higher PRNT50 titres against the panel of SARS-CoV-2 isolates than vaccinated individuals who were uninfected (Fig. 4a, b). With the exception of virus isolates from lineages A, B.1.526a–c and R.1, which affected neutralization the least, all other assayed isolates had a significantly higher NAb response in vaccinated individuals who were previously infected (Fig. 4b); only virus isolates with the E484K and N501Y/T mutations still significantly reduced neutralization (Fig. 4a). For example, the lineage B.1.351b isolate (E484K and N501Y) decreased neutralization titres by 13.2-fold (compared with lineage A) in vaccinated individuals who were uninfected and by 3.7-fold in vaccinated individuals who were previously infected, whereas B.1.617.2 (L452R) went from 6.9-fold to 1.5-fold, and B.1.1.7a (N501Y) went from 3.4-fold to 0.8-fold decrease in individuals who were uninfected to individuals who were infected, respectively (Fig. 4a). Thus, our data suggest that plasma neutralization activity against SARS-CoV-2 variants is improved in vaccinated individuals who were previously infected with the virus.

Fig. 4: Comparison of neutralizing activity in vaccinated HCWs who were previously infected or not to SARS-CoV-2.
figure 4

Plasma neutralization titres against ancestral lineage A virus (WA1, USA) and locally circulating variants of concern or interest, and other lineages. Sixteen SARS-CoV-2 variants were isolated from nasopharyngeal swabs of individuals who were infected and an additional B.1.351 isolate was obtained from BEI. Neutralization capacity between vaccinated participants who were previously infected or not with SARS-CoV-2 is also shown, and was accessed using plasma samples from vaccinated participants, 28 days after the second dose of the SARS-CoV-2 vaccine at the experimental sixfold serial dilutions (from 1:3 to 1:2,430). a, Neutralization titre among vaccinated individuals. Significance was assessed by one-way ANOVA corrected for multiple comparisons using Dunnett’s method. Neutralization capacity to the variants was compared with the neutralization capacity against the ancestral strain. The bars represent mean values ± standard deviations; the dotted line indicates the mean value of PRNT50 to the ancestral strain. Variants were grouped giving specific spike mutations and are coloured accordingly. b, Comparison of the neutralization titre among vaccinated participants who were previously infected or not to SARS-CoV-2. Significance was accessed using unpaired two-tailed t-test. The bars represent mean values ± standard deviations. (−) Vaccinated, uninfected: n = 17; (+) Vaccinated, previously infected: n = 15. Each dot represents a single individual. ****P < 0.0001, ***P < 0.001 **P < 0.01 and *P < 0.05.

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Discussion

Human NAbs against SARS-CoV-2 can be categorized as belonging to four classes on the basis of their target regions on the RBD. Although the RBD is immunodominant, there is evidence for a substantial role of other spike regions in antigenicity, most notably the NTD supersite22,23,24. These antibodies target epitopes that are closely associated with NTD and RBD residues L18 and ΔH69/V70, and K417, L452, S477, T478, E484 and N501. Previous studies using pseudovirus constructs reported a significant effect of single spike gene amino acid substitutions, including S477N and E484K located at the RBD–ACE2 interface, in the neutralization activity of plasma from vaccinated individuals1,2,3,4,5,25.

Using a large panel of genetically diverse authentic SARS-CoV-2 isolates, we found that lineages with E484K and N501Y/T led to the most severe decreases in mRNA vaccine-induced neutralization (more than tenfold in vaccinated individuals who were previously uninfected). This group includes B.1.351 (Beta) and P.1 (Gamma), further supporting their importance in regards to vaccines. We also found that a generic lineage B.1 isolate with E484K and N501T, and a rare B.1.1.7 (Alpha) isolate with E484K (also with the common N501Y mutation) have similar effects on neutralization as B.1.351 and P.1. While the combinations of mutations in the B.1 and B.1.1.7 with E484K isolates probably do not increase transmissibility, the additive effects of these two mutations support that surveillance programmes should track all viruses with E484K and N501Y/T mutations in addition to variants of concern and/or interest.

While we estimate that the L452R mutation has the greatest individual effect on neutralization, lineages with this mutation, including B.1.617.2 (Delta), are less concerning for NAb escape than the E484K–N501Y/T combination. From our data, we expect that most fully vaccinated individuals will be protected against B.1.617.2, and that the rise in vaccine-breakthroughs associated with this variant are more probably associated with its high transmissibility26,27,28,29.

The discrepancies of our results compared with other studies, including ref. 6, may point to the importance of using fully intact, authentic virus for neutralization assays to detect the effects of epistasis among virus mutations on neutralization assays. Nevertheless, it remains possible that additional factors also contribute to some of the discrepancies between our observations and those of previous studies, including the presence of additional mutations in the membrane and envelope, as well as the composition of our cohorts, predominantly young white women. Differences between cohorts could also account for subtle differences in T cell responses observed in our study versus the one recently reported by ref. 16 and ref. 17. While we observed decreased cross-reactivity of spike-reactive CD8+ T cells against P.1 spike peptides, the above studies found that T cell responses are largely preserved against variants16,17. In addition to cohort composition, we used overlapping peptide pools in our assays, and it remains possible that our T cell assay failed to detect under-represented T cell clones affected by variant sequences when sampled in the presence of the majority of conserved peptides. Furthermore, ref. 16 used an AdV1.26 vaccine, whereas in this study and ref. 17, mRNA vaccines were used. Overall, our data point to a necessity of active monitoring of T cell reactivity in the context of SARS-CoV-2 evolution.

The magnitude of the antibody titres in patients with COVID-19 following natural infection has been directly correlated with length of infection and severity30. Here we found that vaccinated individuals who were previously infected display an increased resilience in antibody responses against both ‘single’ and a combination of substitutions in the RBD region, which otherwise severely decreased neutralization activity of vaccinated individuals who were uninfected. Our observations of the effect of pre-existing immunity in vaccinated individuals on their ability to neutralize variants could be explained by the time window between the initial exposure (infection) and vaccination. Moreover, on the basis of timing of previous infection (before the emergence of tested variants) and confirmation by sequencing (three previous infections with B.1.3), we believe that the virus lineage of the infection probably did not have a major effect on our findings. Our observations provide an important rationale for worldwide efforts in characterizing the contribution of pre-existing SARS-CoV-2 immunity to the outcome of various vaccination strategies. Along with recently introduced serological tests31, such studies could inform evidence-based risk evaluation, patient monitoring, adaptation of containment methods, and vaccine development and deployment. Finally, these findings suggest that an additional third dose of a vaccine may be beneficial to confer higher protection against SARS-CoV-2 lineages such as B.1.351 and P.1.

Methods

Ethics statement

This study was approved by the Yale Human Research Protection Program Institutional Review Board (IRB protocol ID 2000028924). Informed consent was obtained from all enrolled vaccinated HCWs. The IRB from the Yale University Human Research Protection Program determined that the RT–qPCR testing and sequencing of de-identified remnant COVID-19 clinical samples conducted in this study were not research involving human participants (IRB protocol ID 2000028599).

HCW volunteers

Forty HCW volunteers from the Yale-New Haven Hospital were enrolled and included in this study. The volunteers received the mRNA vaccine (Moderna or Pfizer) between November 2020 and January 2021. Vaccinated donors were stratified in two major groups, previously infected with SARS-CoV-2 (recovered) or uninfected (naive), confirmed by RT–qPCR (10 April 2020 to 31 December 2020) and serology (2 April 2020 to 11 March 2020). None of the participants experienced serious adverse effects after vaccination. HCWs were followed serially post-vaccination. Plasma and PBMC samples were collected at baseline (previous to vaccination), 7 and 28 after the first vaccination dose, and 7, 28 and 70 days after the second vaccination dose. Demographic information was aggregated through a systematic review of the electronic health record (EHR) and was used to construct Extended Data Table 1. The clinical data were collected using EPIC EHR May 2020 and REDCap 9.3.6 software. Blood acquisition was performed and recorded by a separate team. Clinical information and time points of collection information of vaccinated HCWs were not available until after processing and analysing raw data by flow cytometry and ELISA. ELISA, neutralizations and flow cytometry analyses were blinded.

Isolation of plasma and PBMCs

Whole blood was collected in heparinized CPT blood vacutainers (BDAM362780, BD) and kept on gentle agitation until processing. All blood was processed on the day of collection in a single step standardized method. Plasma samples were collected after centrifugation of whole blood at 600g for 20 min at room temperature without a break. The undiluted plasma was transferred to 15-ml polypropylene conical tubes, and aliquoted and stored at −80 °C for subsequent analysis. The PBMC layer was isolated according to the manufacturer’s instructions. Cells were washed twice with PBS before counting. Pelleted cells were briefly treated with ACK lysis buffer for 2 min and then counted. Percentage viability was estimated using standard trypan blue staining and an automated cell counter (AMQAX1000, Thermo Fisher). PBMCs were stored at −80 °C for subsequent analysis.

SARS-CoV-2-specific antibody measurements

ELISAs were performed as previously described32. In short, Triton X-100 and RNase A were added to serum samples at final concentrations of 0.5% and 0.5 mg/ml, respectively, and incubated at room temperature for 30 min before use, to reduce risk from any potential virus in serum. MaxiSorp plates (96 wells; 442404, Thermo Scientific) were coated with 50 μl per well of recombinant SARS-CoV-2 STotal (SPN-C52H9-100 μg, ACROBiosystems), S1 (S1N-C52H3-100 μg, ACROBiosystems), RBD (SPD-C52H3-100 μg, ACROBiosystems) and the nucleocapsid protein (NUN-C5227-100 μg, ACROBiosystems) at a concentration of 2 μg/ml in PBS and were incubated overnight at 4 °C. The coating buffer was removed, and plates were incubated for 1 h at room temperature with 200 μl of blocking solution (PBS with 0.1% Tween-20 and 3% milk powder). Plasma was diluted serially at 1:100, 1:200, 1:400 and 1:800 in dilution solution (PBS with 0.1% Tween-20 and 1% milk powder), and 100 μl of diluted serum was added for 2 h at room temperature. Human anti-spike (SARS-CoV-2 human anti-spike (AM006415) (91351, Active Motif) and anti-nucleocapsid SARS-CoV-2 human anti-nucleocapsid (1A6) (MA5-35941, Active Motif) were serially diluted to generate a standard curve. Plates were washed three times with PBS-T (PBS with 0.1% Tween-20) and 50 μl of HRP anti-human IgG antibody (1:5,000; A00166, GenScript) diluted in dilution solution added to each well. After 1 h of incubation at room temperature, plates were washed six times with PBS-T. Plates were developed with 100 μl of TMB Substrate Reagent Set (555214, BD Biosciences) and the reaction was stopped after 5 min by the addition of 2 N sulfuric acid. Plates were then read at a wavelength of 450 nm and 570 nm.

T cell stimulation

For the in vitro stimulation, PBMCs were stimulated with HLA class I and HLA-DR peptide pools at the concentration of 1–10 μg ml−1 per peptide and cultured for 7 days. On day 0, PBMCs were thawed, counted and plated in a total of 5–8 × 105 cells per well in 200 ul of RPMI 1640 medium (Gibco) supplemented with 1% sodium pyruvate (NEAA), 100 U/ml penicillin–streptomycin (Biochrom) and 10% FBS at 37 °C and 5% CO2. On day 1, cells were washed and the stimulation was performed with: PepMix SARS-CoV-2 spike glycoprotein pool 1 and pool 2 (GenScript), PepMix P.1 SARS-CoV-2 spike glycoprotein pool 1 and pool 2 (JPT) and PepMix SARS-CoV-2 nucleocapsid protein (JPT). Stimulation controls were performed with PBS (unstimulated). Peptide pools were used at 1 μg ml−1 per peptide. Incubation was performed at 37 °C, 5% CO2 for 6 days. On day 6, cells were restimulated with 10 μg ml−1 per peptide and subsequently incubated for 12 h, with the last 6 h being in the presence of 10 μg ml−1 brefeldin A (Sigma-Aldrich). Following this incubation, cells were washed with PBS 2 mM EDTA and prepared for analysis by flow cytometry.

Flow cytometry

Antibody clones and vendors were as follows: BB515 anti-hHLA-DR (G46-6, 1:400; BD Biosciences), BV605 anti-hCD3 (UCHT1, 1:300; BioLegend), BV785 anti-hCD19 (SJ25C1, 1:300; BD Biosciences), BV785 anti-hCD4 (SK3, 1:200; BioLegend), APCFire750 or BV711 anti-hCD8 (SK1, 1:200; BioLegend), AlexaFluor 700 anti-hCD45RA (HI100, 1:200; BD Biosciences), PE anti-hPD1 (EH12.2H7, 1:200; BioLegend), APC or PE-CF594 anti-hTIM3 (F38-2E2, 1:50; BioLegend), BV711 anti-hCD38 (HIT2, 1:200; BioLegend), BB700 anti-hCXCR5 (RF8B2, 1:50; BD Biosciences), PE-CF594 anti-hCD25 (BC96, 1:200; BD Biosciences), AlexaFluor 700 anti-hTNFa (MAb11, 1:100; BioLegend), PE or APC/Fire750 anti-hIFNγ (4S.B3, 1:60; BioLegend), FITC anti-hGranzymeB (GB11, 1:200; BioLegend), BV785 anti-hCD19 (SJ25C1, 1:300; BioLegend), BV421 anti-hCD138 (MI15, 1:300; BioLegend), AlexaFluor700 anti-hCD20 (2H7, 1:200; BioLegend), AlexaFluor 647 anti-hCD27 (M-T271, 1:350; BioLegend), PE/Dazzle594 anti-hIgD (IA6-2, 1:400; BioLegend), Percp/Cy5.5 anti-hCD137 (4B4-1, 1:150; BioLegend) and PE anti-CD69 (FN-50, 1:200; BioLegend), and APC anti-hCD40L (24–31, 1:100; BioLegend). In brief, freshly isolated PBMCs were plated at 1–2 × 106 cells per well in a 96-well U-bottom plate. Cells were resuspended in Live/Dead Fixable Aqua (Thermo Fisher) for 20 min at 4 °C. Following a wash, cells were blocked with Human TruStan FcX (BioLegend) for 10 min at room temperature. Cocktails of desired staining antibodies were added directly to this mixture for 30 min at room temperature. For secondary stains, cells were first washed and supernatant aspirated; then to each cell pellet, a cocktail of secondary markers was added for 30 min at 4 °C. Before analysis, cells were washed and resuspended in 100 μl 4% PFA for 30 min at 4 °C. Following this incubation, cells were washed and prepared for analysis on an Attune NXT (Thermo Fisher). Data were analysed using FlowJo software version 10.6 software (Tree Star). The specific sets of markers used to identify each subset of cells are summarized in Extended Data Fig. 4.

Cell lines and virus

TMPRSS2-VeroE6 kidney epithelial cells were cultured in DMEM supplemented with 1% sodium pyruvate (NEAA) and 10% FBS at 37 °C and 5% CO2. The cell line was obtained from the American Type Culture Collection and tested negative for contamination with mycoplasma. SARS-CoV-2 lineage A (USA-WA1/2020) was obtained from BEI Resources (no. NR-52281) and was amplified in TMPRSS2-VeroE6. Cells were infected at a multiplicity of infection of  0.01 for 3 days to generate a working stock, and after incubation, the supernatant was clarified by centrifugation (450g for 5 min), and filtered through a 0.45-µm filter. The pelleted virus was then resuspended in PBS and aliquoted for storage at −80 °C. Viral titres were measured by standard plaque assay using TMPRSS2-VeroE6. In brief, 300 µl of serial fold virus dilutions were used to infect Vero E6 cells in MEM supplemented with NaHCO3, 4% FBS and 0.6% Avicel RC-581. Plaques were resolved at 48 h post-infection by fixing in 10% formaldehyde for 1 h followed by 0.5% crystal violet in 20% ethanol staining. Plates were rinsed in water to plaques enumeration. All experiments were performed in a biosafety level 3 laboratory with approval from the Yale Environmental Health and Safety office.

SARS-CoV-2 variant sequencing and isolation

SARS-CoV-2 samples were sequenced as part of the Yale SARS-CoV-2 Genomic Surveillance Initiative’s weekly surveillance programme in Connecticut, USA33. Lineages were sequenced and isolated as previously described34. In brief, nucleic acid was extracted from de-identified remnant nasopharyngeal swabs and tested with our multiplexed RT–qPCR variant assay to select samples with a N1 cycle threshold value of 35 or lower for sequencing35,36. Libraries were prepared with a slightly adjusted version of the Illumina COVIDSeq Test RUO version. The Yale Center for Genome Analysis sequenced pooled libraries of up to 96 samples on the Illumina NovaSeq (paired-end 150). Data were analysed and consensus genomes were generated using iVar (version 1.3.1)37. Variants of interest and concern, lineages with mutations of concern (E484K), as well as other lineages as controls were selected for virus isolation. In total, 16 viruses were isolated belonging to 12 lineages (Extended Data Fig. 3, Extended Data Table 2). In addition, ancestral lineage A virus and lineage B.1.351 virus were obtained from BEI.

Samples selected for virus isolation were diluted 1:10 in DMEM and then filtered through a 45-µm filter. The samples were tenfold serially diluted from 1:50 to 1:19,531,250. The dilution was subsequently incubated with TMPRSS2-Vero E6 in a 96-well plate and adsorbed for 1 h at 37 °C. After adsorption, replacement medium was added, and cells were incubated at 37 °C for up to 5 days. Supernatants from cell cultures with cytopathic effect were collected, frozen, thawed and subjected to RT–qPCR. Fresh cultures were inoculated with the lysates as described above for viral expansion. Viral infection was subsequently confirmed through reduction of cycle threshold values in the cell cultures with the multiplex variant qPCR assay. Expanded viruses were resequenced following the same method as described above and genome sequences were uploaded to GenBank (Supplementary Data Table 2), and the aligned consensus genomes are available on GitHub (https://github.com/grubaughlab/paper_2021_Nab-variants). Nextclade v1.5.0 (https://clades.nextstrain.org/) was used to generate a phylogenetic tree (Extended Data Fig. 3) and to compile a list of amino acid changes in the virus isolates as compared with the Wuhan-Hu-1 reference strain (Extended Data Table 2). Key spike amino acid differences were identified based on the outbreak.info mutation tracker20.

Neutralization assay

Sera from vaccinated HCWs were isolated as described before and then heat treated for 30 min at 56 °C. Sixfold serially diluted plasma, from 1:3 to 1:2,430 were incubated with SARS-CoV-2 variants, for 1 h at 37 °C. The mixture was subsequently incubated with TMPRSS2-VeroE6 in a 12-well plate for 1 h, for adsorption. Then, cells were overlayed with MEM supplemented with NaHCO3, 4% FBS and 0.6% Avicel mixture. Plaques were resolved at 40 h post-infection by fixing in 10% formaldehyde for 1 h followed by staining in 0.5% crystal violet. All experiments were performed in parallel with sera from baseline controls, in an established viral concentration to generate 60–120 plaques per well.

Statistical analysis

All analyses of patient samples were conducted using GraphPad Prism 8.4.3, JMP 15 and R 3.4.3. Multiple group comparisons were analysed by running parametric (ANOVA) statistical tests. Multiple comparisons were corrected using Tukey’s and Dunnett’s tests as indicated in the figure legends. For the comparison between stable groups, two-sided unpaired t-test was used for the comparison. The effect of spike mutations was assessed using a linear mixed model with an outcome of log-transformed PRNT50 and random effects accounting for each individual participant. This was done using the ‘lme4’ package in R 4.0.1 (ref. 38).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.