Broad humoral and cellular immunity elicited by one-dose mRNA vaccination 18 months after SARS-CoV-2 infection

Serum and peripheral blood mononuclear cell (PBMC) samples were prospectively collected from COVID-19 convalescent individuals who received one dose of an mRNA vaccine either within 6 months (Conv6mVx1 group) or around 18 months (Conv18mVx1 group) of a COVID-19 diagnosis. Additional serum samples were collected from COVID-19 convalescent individuals who received two doses of an mRNA vaccine (Conv6mVx2 and Conv18mVx2 groups) and from healthy healthcare workers (NonConvVx0, NonConvVx1, and NonConvVx2 groups). The post-vaccination sample was collected 2−4 weeks after each vaccination. All serum samples were stored at −80 °C until use in the assays.

All serum samples were examined for IgG-binding activity against the receptor-binding domain (RBD) of SARS-CoV-2 WT and the Alpha, Beta, Delta, and Omicron variants (RBD_wt, RBD_α, RBD_β, RBD_γ, RBD_δ, and RBD_ο). Samples from the Conv6mVx1, Conv18mVx1, Conv18mVx2, NonConvVx1, and NonConvVx2 groups were additionally assessed for neutralizing activity, and PBMCs from the Conv6mVx1 and Conv18mVx1 groups were assessed for cell-mediated immune responses against WT SARS-CoV-2 and the Delta variant.

All COVID-19 convalescent individuals had been laboratory-confirmed with real-time reverse transcription polymerase chain reaction and admitted to the Seoul National University Hospital or a community treatment center and had mild or asymptomatic disease with no oxygen requirement [11, 12]. The Institutional Review Board of Seoul National University Hospital approved the study (IRB nos. 2009-168-1160 and 2102-032-1193). Written informed consent was obtained from all participants according to the Declaration of Helsinki.

Genes encoding RBD of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants were cloned in-frame into the pcDNA3.4-SARS-CoV-2-spike RBD-his using Gibson Assembly cloning (NEB, Ipswich, MA, USA) [13]. SARS-CoV-2 antigens were produced in Expi293 cells (Thermo Fisher Scientific) and his–tagged SARS-CoV-2 antigens protein was purified using Ni-NTA agarose resin (Thermo Fisher Scientific) affinity chromatography, as described previously [13].

After 5 days of transfection, the supernatant was collected and passed over the Ni-NTA agarose resin column three times. First, the column was washed with 100 mL of 1× PBS to remove nonspecific bound proteins. Then, 3 mL of elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole) was added to elute the bound proteins. Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel (Additional file 1: Supplementary Fig. 1).

For measuring the binding activity of serum antibody (Ab) against each SARS-CoV-2 antigen (RBD_wt, RBD_α, RBD_β, RBD_γ, RBD_δ, and RBD_ο), 100 ng per well of each antigen was coated on a 96-well polystyrene enzyme-linked immunosorbent assay (ELISA) plate (Thermo Fisher Scientific) for overnight at 4 °C. After blocking with 1× PBS (pH 7.4) containing 3% bovine serum albumin (BSA) for 1 h at room temperature, the plate was washed four times with the PBST buffer (PBS with 0.05% Tween 20). The diluted plasma (1:50) was added and incubated at room temperature for 1 h.

After washing with the PBST buffer four times to detect IgG level, mouse anti-human IgG Fc Ab–conjugated with HRP (1:12,000, Arigobio, Hsinchu, Taiwan) was added and incubated for 1 h at room temperature. After washing four times with the PBST buffer, 50 μL of 3,3′,5,5′-tetramethylbenzidine substrate was added per well (Thermo Fisher Scientific) and then 50 μL of 2 M H₂SO₄ was added to neutralize. Finally, the absorbance at 450 nm was measured using the Infinite 200 PRO NanoQuant microplate readers (Tecan Trading AG, Männedorf, Switzerland).

Vero E6 (ATCC# CRL-1586) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone, South Logan, Utah, USA) and 1% antibiotics (Cytiva, South Logan, Utah, USA) [14]. SARS-CoV-2 isolates, SARS-CoV-2/human/KOR/KCDC03-NCCP 43326/2020 (GenBank ID. MW466791.1, S clade) [15], and SARS-CoV-2/human/KOR/NCCP 43390/2021 (GenBank ID. OL966962.1, Delta variant) were obtained from the National Culture Collection for Pathogens, Korea Disease Control, and Prevention Agency.

Vero E6 cells were inoculated with SARS-CoV-2 isolate at 1 multiplicity of infection (MOI), and fresh DMEM with 2% FBS and 1% antibiotics was added. At 48 h after infection, the supernatants of virus-infected cells were harvested and stored at −80 °C. Viral titers were determined by plaque assay on Vero E6 cells as described previously [16, 17]. All work with SARS-CoV-2 was conducted in biosafety level 3 facilities in accordance with the guidelines of and approved by the Institutional Biosafety Committee of Yonsei University Health System.

Vero E6 cells were seeded into 12-well plates at 24 h before infection. Cells were inoculated with ten-fold serial dilutions of SARS-CoV-2 samples and adsorbed at 37 °C in a 5% CO₂ incubator for 1 h, rocking every 15 min. After virus adsorption, inoculums were removed from cells and then 1.5 ml of 1% agarose (Lonza 50101, Rockland, ME, USA) prepared in DMEM with 2% FBS and 1% antibiotics was added. To stain plaques performed with an agarose overlay, 4% paraformaldehyde (Biosesang, Gyeonggi-do, Republic of Korea) was added on top of the agarose and incubated for 24 h at 4 °C. The agarose plug was removed, and the fixed monolayer was stained with 0.5% crystal violet in 20% methanol. The monolayers were washed with topwater. The number of plaques was counted. Viral titers were calculated in plaque-forming units (PFU) per milliliter.

The focus reduction neutralization test (FRNT) was performed by a slightly modified version of a method described previously [18, 19]. A day before viral infection, Vero E6 cells were seeded into 96-well tissue culture plates (1 × 10⁴ cells/well) and incubated at 37 °C for 24 h. Heat-inactivated serum samples were centrifuged at 16,000×g for 20 s at 4 °C. The serum specimens were serially diluted with serum-free-DMEM in 96-well U bottom plates and mixed with an equal volume of SARS-CoV-2 viral stock (600 PFU/well) following 1 h incubation at 37 °C with 5% CO₂. Subsequently, the immune complexes formed by serum and SARS-CoV-2 were overlaid on top of Vero E6 cells and incubated at 37 °C with 5% CO₂ for 1 h. After virus adsorption, the immune complexes were removed and replaced by DMEM with 1% methylcellulose (Sigma-Aldrich, Saint Louis, MO, USA) and 2% FBS. The infected cells were incubated at 37 °C with 5% CO₂ for 17 h and then the media containing methylcellulose were washed out with PBS. The cells were fixed with 2% paraformaldehyde (Biosesang, Gyeonggi-do, Republic of Korea) at 4 °C for 24 h and washed out with PBS. The fixed cells were permeabilized with 0.1% saponin (Sigma-Aldrich) in PBS containing 0.1% BSA for 20 min at room temperature and incubated with a rabbit anti-SARS-CoV/CoV-2 nucleocapsid Ab (Sino Biological, Beijing, China) for 1 h at room temperature. Following washing with PBS, the cells were incubated with a goat anti-rabbit IgG HRP-conjugated secondary Ab for 1 h at room temperature. The cells were incubated with KPL SureBlue Peroxidase Substrate (Seracare, MA, USA) at room temperature and washed with PBS. The cell control (CC) with only cells and the virus control (VC) with virus and cells were set up inonach plate. The foci were visualized and analyzed using an ImmunoSpot reader (Cellular Technology Limited, Cleveland, USA). The percentage of neutralization was calculated as follows: [1 – (the number of foci for sample – the number of foci for CC)/(the number of foci for VC – the number of foci for CC)] × 100. The inhibitory concentration that neutralizes 50% (IC₅₀) of SARS-CoV-2 infection was calculated using a 4-parameter nonlinear regression of GraphPad Prism 9 software.

Pseudovirus expressing the SARS-CoV-2 S protein was produced as described previously [20]. HIV-1 NL4-3 ΔEnv Vpr Luciferase Reporter Vector from Dr. Nathaniel Landau [21, 22] and GP-pCAGGS with SARS-CoV-2 S were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. These plasmids were co-transfected into 293T cells. Forty-eight hours later, SARS-CoV-2 pseudovirus-containing supernatants were harvested, filtered through a 0.45-μm hydrophilic polyethersulfone syringe filter (Pall Corporation, Port Washington, NY, USA), and concentrated by ultracentrifugation at 25700 rpm for 3 h at 4 °C in a Beckman SW32Ti swinging bucket rotor lined with a Beckman thin wall polypropylene centrifuge tube (Beckman Coulter, High Wycombe, UK). The number of infectious virus particles was quantified using the Median Tissue Culture Infectious Dose (TCID₅₀) assay [23]. 1300 TCID₅₀/mL of pseudovirus was mixed with 6 serially diluted serum samples from the COVID-19 patients at 37 °C for 1h. Then, the mixtures were transferred to 96-well plates containing monolayers of 293T cells expressing ACE2 (hACE2-293T) (Takara Bio Inc., Japan). After incubation for 48 h, the cells were harvested with 100 μL of 1× luciferase cell culture lysis reagent and analyzed for luciferase activity by the addition of luciferase substrate using GloMax® discover (all from Promega, Madison, WA, USA). Percent neutralization was normalized as 100% neutralization of uninfected cells and 0% neutralization of pseudovirus-only infected cells.

The whole blood was collected in heparin-containing Vacutainer tubes (Becton Dickinson). PBMCs were purified after blood collection using Ficoll-Hypaque (1.077 g/mL; GE Healthcare Life Sciences, Piscataway, NJ, USA). The PBMCs were then stored in liquid nitrogen in a serum-free cryopreservation medium (Cellbanker 2, Zenoaq) until examined [24]. Cells were cultured in complete RPMI-1640 containing 10% FBS and 1× penicillin/streptomycin (Thermo Fisher Scientific) and stimulated as follows.

After thawing, 1×10⁶ PBMCs/mL were immediately stimulated with 0.06 nmol/mL of PepTivator® SARS-CoV-2 Prot_S Complete, PepTivator® SARS-CoV-2 Prot_S B.1.617.2 WT reference, or PepTivator® SARS-CoV-2 Prot_S B.1.617.2 Mutation Pool (all from Miltenyi Biotec, Bergisch Gladbach, Germany) for 24 h. A CEF peptide pool (Mabtech AB, Hamburg, Germany) and medium alone were used as a positive and negative control. Anti-human CD28/CD49d Abs for co-stimulation (clone L293/L25) and Brilliant Blue 515–anti-human CD4 (clone RPA-T4) Ab were added concomitantly with the antigens. Cells were treated with BD GolgiStop® (monensin) and BD GolgiPlug® (brefeldin A) for the final 4 h of the antigen stimulation.

After stimulation, dead cells were stained with LIVE/DEAD (Thermo Fisher Scientific). Cells were permeabilized and incubated with Brilliant Violet (BV) 510–anti-human CD3 (clone CHT1), peridinin chlorophyll protein complex–anti-human CD8 (clone SK1), phycoerythrin–indotricarbocyanine (Cy7)–anti-human interferon-γ (IFN-γ, clone B27), allophycocyanin–anti-human interleukin-2 (IL-2) (clone 5344.111), phycoerythrin–anti-human tumor necrosis factor-α (TNF-α) (clone Mab11), BV421–anti-human IL-4 (clone MP4-25D2), BV605–anti-human CD69 (clone FN50), and BUV395–anti-human CD137 (clone 4B4-1) Abs (all from BD Biosciences). BD Horizon Brilliant Stain Buffer (BD Biosciences) was added to each sample. Unstimulated cells and compensation beads (UltraComp eBeads, Thermo Fisher Scientific) were used in every experiment for compensation. Flow cytometry was performed using the FACSymphony (BD Biosciences) with a target event count of 1,000,000 cells. The flow cytometry results were analyzed using FlowJo software (version 10.7.1; TreeStar). A representative gating strategy is shown in Additional file 2: Supplementary Fig. 2.

To account for nonspecific cytokine production, the percentage of the target population in the unstimulated specimens was subtracted from the percentage in the stimulated cells [25]. The frequencies of SARS-CoV-2-specific activation-induced marker⁺ (AIM⁺) T cells (CD69⁺CD137⁺ CD4⁺ T cells or CD8⁺ T cells) [26] or cytokine-producing cells (among CD137⁺CD4⁺ or CD137⁺CD8⁺ T cells) were evaluated.

The data are presented as the mean ± standard error of the mean (SEM) and as dot plots. A Wilcoxon signed-rank test (paired samples from uninfected healthcare workers) or Mann-Whitney U test (unpaired samples from COVID-19 convalescent individuals) was performed to compare immune responses between two groups. The Benjamini-Hochberg method was used to account for multiple comparisons between uninfected and infected groups. P < 0.05 was considered indicative of statistical significance. All statistical analyses were two-tailed and performed using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). All graphs were drawn using GraphPad Prism 9.

We enrolled 10, 5, 18, and 10 individuals in the Conv6mVx1, Conv6mVx2, Conv18mVx1, and Conv18mVx2 groups, respectively. In addition, serum samples were collected from 10 uninfected healthcare workers in the NonConvVx groups. All COVID-19-convalescent individuals had a SARS-CoV-2 infection before the Delta surge in Korea [27].

The median age was 45, 43, 27, 30, and 33 years in the Conv6mVx1, Conv6mVx2, Conv18mVx1, Conv18mVx2, and NonConvVx groups, respectively. None of the participants had significant concurrent medical conditions. Among the convalescent individuals, none had received specific treatment for COVID-19, and none had a known history of re-exposure to SARS-CoV-2 after the initial infection. Detailed information on the type of mRNA vaccine, the interval between the COVID-19 diagnosis and vaccination, and the interval between each vaccination and sample collection are reported in Additional file 3: Supplementary Table 1.

All serum samples were examined for serum IgG binding activity using ELISA against the RBD of SARS-CoV-2 WT (RBD_wt), Alpha (RBD_α), Beta (RBD_β), Gamma (RBD_γ), Delta (RBD_δ), and Omicron (RBD_ο) variants. Although the NonConvVx group had a significant humoral response by the second vaccination, humoral responses in the Conv6mVx and Conv18mVx groups were readily boosted by the first vaccination (Fig. 1a).

Serum IgG of Conv18mVx1 showed equivalent binding activities for RBD_wt, RBD_α, RBD_β, RBD_δ, and RBD_ο comparing with those of the Conv6mVx1 groups (Fig. 1a). The serum IgG RBD_γ-binding activities were significantly stronger in the Conv18mVx1 group (median [interquartile range, IQR], 3.39 [3.30−3.48]) than in the Conv6mVx1 group (median [IQR], 3.07 [2.77−3.36]) (adjusted P = 0.047). Interestingly, the second vaccination did not improve the humoral IgG response to any of the RBD variants, either in the Conv6mVx or Conv18mVx group, and the response to the Beta variant declined (median [IQR], 2.95 [2.75−3.46] in the Conv6mVx1 group vs. 2.34 [1.62−2.71] in the Conv6mVx2 group, adjusted P = 0.032). The binding activity of serum IgG from the Conv18mVx1 group was significantly lower for RBD_ο than the other RBD variants (Additional file 4: Supplementary Fig. 3a), as previously reported [28].

Neutralizing activity against SARS-CoV-2 WT and the Delta variant was investigated in FRNT₅₀ using serum samples from the Conv6mVx1, Conv18mVx1, Conv18mVx2, NonConvVx1, and NonConvVx2 groups. Log-transformed FRNT₅₀ values were higher in the Conv18mVx1 than the Conv6mVx1 group (median [IQR] for WT, 2.72 [2.49−3.52] in the Conv6mVx1 group vs. 3.66 [3.25−4.24] in the Conv18mVx1 group, adjusted P = 0.013; for the Delta variant, 2.78 [1.99−3.15] in the Conv6mVx1 group vs. 3.29 [3.08−3.66] in the Conv18mVx1 group, adjusted P = 0.013) (Fig. 1b). A pseudovirus neutralization test against WT SARS-CoV-2 yielded similar results, where area under the curve (AUC) values were higher in the Conv18mVx1 than the Conv6mVx1 group (median [IQR], 196.40 [179.63−238.30] in the Conv6mVx1 group vs. 271.50 [248.88−289.15] in the Conv18mVx1 group, adjusted P < 0.001) (Fig. 1c).

Similar to the results of the serum IgG-binding activity assay, the second dose of an mRNA vaccine did not induce an additional response in the Conv18mVx group. Still, it significantly raised the FRNT₅₀ and AUC values in the uninfected group. The neutralizing activity of the serum samples in the Conv18mVx1 group, as measured in a live virus neutralization assay, was considerably lower against the Delta variant than against WT SARS-CoV-2 (Additional file 4: Supplementary Fig. 3b)

T cell-mediated immune responses against SARS-CoV-2 WT, and the Delta variant in the Conv6mVx1 and Conv18mVx1 groups were assessed using flow cytometric analysis. PBMCs were separately stimulated by three different SARS-CoV-2 antigens to examine the response to the whole spike peptide pool and matched WT and Delta spike mutation peptide pools. T cell-mediated immune responses were measured by the expression by AIM (CD69⁺CD137⁺ in both CD4⁺ and CD8⁺ T cells) or cytokines (IL-2, IL-4, TNF-α, and IFN-γ in both CD137⁺CD4⁺ and CD137⁺CD8⁺ T cells.

The proportions of AIM⁺ CD4⁺ T cells against WT whole spike protein did not differ between the Conv6mVx1 and Conv18mVx1 groups (Fig. 2a), while those of CD8⁺ T cells were significantly higher in the Conv6mVx1 than in the Conv18mVx1 group (median [IQR], 0.29% [0.13−0.55%] vs. 0.08% [0.02−0.13%], P = 0.003). No significant differences were observed in the proportions of AIM⁺ T cells against matched WT and Delta mutation peptide pools (Fig. 2b, c).

The proportions of cytokine (IL-2, IL-4, TNF-α, or IFN-γ)-producing CD4⁺ or CD8⁺ T cells in the Conv6mVx1 and Conv18mVx1 groups also showed no significant difference (Additional file 5: Supplementary Fig. 4a and 4b). In addition, there was no significant difference in the proportions of AIM⁺ CD4⁺ or CD8⁺ T cells against WT and the Delta variant from the Conv18mVx1 group (Additional file 4: Supplementary Fig. 3c), as previously reported in the literature [6].