Vaccination impairs de novo immune response to omicron breakthrough infection, a precondition for the original … – Nature.com

Study cohort

A total of 106 individuals were included in this study. 87 were recruited by the occupational healthcare department of the University Hospital Bonn and 19 by the Emergency Medicine department of the University Gttingen in Germany. The first contact was established by telephone after which a written invitation and a consent form were sent to each participant. Individuals were divided into three groups according to their histories of exposure to SARS-CoV-2 antigens: individuals who had received three mRNA (encoding wild-type spike protein) vaccine doses and subsequently recovered from an omicron breakthrough infection (Vacc+O-Inf, n=37), individuals who received three mRNA (encoding wild-type spike protein) vaccine doses and were not infected with SARS-CoV-2 (Vacc, n=41), and individuals that did not get vaccinated but were infected with omicron (O-Inf, n=28). Age or sex was not among the selection criteria. Following gender distribution was observed between the groups: 65% females and 35% males for the Vacc+O-Inf group, 66% females and 34% males for the Vacc group, 50% females and 50% males for the O-Inf group, and 57% females and 43% males for the subgroup of 7 O-Inf individuals with available PBMC samples. No significant differences in age distribution were observed between the groups (mean yearsSD for O-Inf, Vacc+O-Inf, Vacc groups and a subgroup of 7 O-Inf individuals with available PBMC samples respectively: 5021, 4015, 4714, 4413). SARS-CoV-2 infections were confirmed by RT-PCR. During the period of sample collection, the prevalence of omicron variants was >99% as assessed by sentinel sequencing. Detailed information on the vaccination, infection, and sampling time points as well as demographic information is provided in supplemental table1. All individuals with omicron SARS-CoV-2 infection did not have previously confirmed SARS-CoV-2 infection. For the Vacc group only individuals without confirmed SARS-CoV-2 infection, and negative nucleocapsid ELISA results were included. Vaccinations of individuals included in this study were performed at the occupational healthcare department of the University Hospital Bonn.

The study was approved by the Ethics Committee of the Medical Faculty of the University of Bonn (ethics approval number 125/21) and the Ethics Committee of University Medical Center Goettingen (ethics approval number 21/06/22). All participants provided written informed consent. No compensation was provided for the participants.

Study participants provided peripheral blood specimens that were centrifuged for 10min at 600g to collect plasma. EDTA plasma was stored until analysis at 80C. PBMC were isolated by density gradient centrifugation using SepMate (Stemcell, 85450) tubes with density gradient medium (Pancoll, PAN-Biotech, P04-60500). The blood was diluted with PBS containing 2% FCS, carefully layered on top of the density gradient medium, and centrifuged at 1200g for 10min. The top layer containing the PBMCs was poured off and washed twice with PBS containing 2% FCS. Washed PBMC were resuspended in FCS containing 10% DMSO and frozen at 80C overnight. For long-term storage, PBMC samples were transferred to liquid nitrogen.

An in-house quantitative ELISA was used for the determination of omicron-SARS-CoV-2-RBD-specific IgG. First, microtiter plates with high binding capacity were coated with 100l of coating buffer (carbonate-bicarbonate buffer, pH=9.6) containing 1g/ml of recombinant omicron SARS-CoV-2 RBD protein (SARS-CoV-2 Spike RBD, His Tag (B.1.1.529/Omicron), Acro Biosystems, SPD-C82E8) and incubated overnight at 4C. After washing with wash buffer (PBS with 0.05% (v/v) Tween-20) plates were blocked (PBS containing 1% (w/v) BSA) to prevent unspecific binding. Cryopreserved EDTA plasma samples were thawed and diluted 400-fold in the blocking buffer. After blocking, plates were washed, incubated with plasma samples, standard dilutions, and negative control (Human IgG Isotype Control, Invitrogen, 12-000-C, 100ng/ml), washed again, and incubated with 100l of HRP-conjugated anti-IgG antibody (Goat anti-Human IgG (Heavy chain) Secondary Antibody, HRP, Invitrogen, A18805) diluted 8000-fold in wash buffer. All incubation steps were performed at 37C for 1hour. Finally, plates were washed and 100l of the substrate solution was added (TMB Chromogen Solution, Life technologies, 002023). The substrate conversion took place at room temperature for 5min until the addition of 50l of 0.2M H2SO4. The optical density at 450nm (OD450) was measured using Synergy 2 Multimode Plate Reader (BioTek). The background-subtracted OD450 readings were interpolated to the standard dilution curve. The positivity cutoff was determined as the mean plus two standard deviations of plasma samples from healthy individuals collected before the COVID-19 outbreak.

An in-house competitive ELISA was used for the determination of IgG specific for the omicron but not wild-type SARS-CoV-2 RBD. Microtiter plates with high binding capacity were coated with 100l of coating buffer (carbonate-bicarbonate buffer, pH=9.6) containing 1g/ml of recombinant omicron SARS-CoV-2 RBD protein (SARS-CoV-2 Spike RBD, His Tag (B.1.1.529/Omicron), Acro Biosystems, SPD-C82E8) or 1g/ml of BSA and incubated overnight at 4C. After washing with wash buffer (PBS with 0.05% (v/v) Tween-20) plates were blocked (PBS containing 1% (w/v) BSA) to prevent unspecific binding. Cryopreserved EDTA plasma samples were thawed and diluted in the blocking buffer. The plasma dilutions were calculated based on the previous measurement of omicron-SARS-CoV-2-RBD-specific IgG to achieve the OD450 of 2. Diluted plasma was then incubated with serial dilutions of wild-type RBD protein (SARS-CoV-2 (COVID-19) S protein RBD, His Tag, Acro Biosystems, SPD-C52H1). A total of 8 dilutions between 1g/ml and 0,002g/ml were measured for each sample. No further technical replicates were performed. Blocked RBD-coated plates were washed, incubated with plasma samples, standard dilutions and negative control (Human IgG Isotype Control, Invitrogen, 12-000-C, 100ng/ml), washed again, and incubated with 100l of HRP-conjugated anti-IgG antibody (Goat anti-Human IgG (Heavy chain) Secondary Antibody, HRP, Invitrogen, A18805) diluted 8000-fold in wash buffer. BSA-coated plates were incubated with three replicates of diluted plasma samples without wild-type RBD and treated equally. All incubation steps were performed at 37C for 1hour. Finally, plates were washed and 100l of the substrate solution was added (TMB Chromogen Solution, Life technologies, 002023). The substrate conversion took place at room temperature for 5min until the addition of 50l of 0.2M H2SO4. The optical density at 450nm was measured using Synergy 2 Multimode Plate Reader (BioTek). The background-subtracted OD450 readings were interpolated to the standard dilution curve. For each plasma sample incubated with wild-type RBD dilution series a scatter plot was generated and a sigmoidal curve was fitted to determine the top (representing the signal from total omicron-RBD-specific IgG) and bottom (representing the signal from omicron-not-wild-type-RBD-specific IgG) plateaus of the curve. GraphPad Prism software version 9.4.1. (681) was used for this purpose. The background signal of the BSA control was then subtracted from the bottom and top plateaus after which the two values were divided to obtain the proportion of omicron-not-wild-type-RBD-specific IgG relative to the total omicron-RBD-specific IgG. This fraction was multiplied with the corresponding quantitative ELISA measurement to obtain the level of omicron-not-wild-type-RBD-specific IgG in plasma.

The plasma neutralization capacity was determined by a plaque reduction neutralization assay. Therefore, plasma was heat-inactivated for 30min at 56C and serially two-fold diluted in OptiPRO SFM (Gibco, 12309-019) cell culture medium. A total of 10 dilutions between 4-fold and 32768-fold were measured for each sample depending on the neutralization capacity of a specimen. No further technical replicates were performed. Each plasma dilution was combined with 80 plaque-forming units of omicron SARS-CoV-2 (B.1.1.529 in OptiPRO SFM (Gibco, 12309-019) serum-free cell culture medium, incubated for 1h at 37C, and added to Vero E6 cells (ATCC, CRL-1586). The cells were seeded in 24-well plates at a density of 1.25105 cells/well 24h earlier. Following 1h incubation at 37C, the inoculum was removed and cells were overlaid with a 1:1 mixture of 1.5% (w/v) carboxymethylcellulose in 2xMEM supplemented with 4% FCS. After incubation at 37C for four days, the overlay was removed and the cells were fixed using a 6% formaldehyde solution. Fixed cells were stained with 1% crystal violet solution revealing the formation of plaques. The number of plaques was plotted against the plasma dilutions, and the half-maximal inhibitory concentration (IC50) was determined using GraphPad Prism software version 9.4.1. (681).

To measure the proportion of neutralizing antibodies that recognize mutated regions of the omicron SARS-CoV-2 surface proteins we developed a competitive plaque reduction neutralization assay. First, plasma was heat-inactivated for 30min at 56C and diluted in OptiPRO SFM (Gibco, 12309-019) serum-free cell culture medium. The plasma dilutions were calculated based on the previous measurement of plasma neutralization capacity against the omicron-SARS-CoV-2 to achieve the 80% neutralization effect. Diluted plasma was then incubated with 12 serial 2-fold dilutions of wild-type SARS-CoV-2 surface proteins, spike (Acro Biosystems, SPN-C52H7), membrane (RayBiotech, YP_009724393) and envelope (Acro Biosystems, ENN-C5128) starting with 10ug/ml and incubated overnight at 4C. Plasma sample dilutions, standard dilutions, and negative controls (media without plasma) were combined with 80 plaque-forming units of omicron SARS-CoV-2 (B.1.1.529) in OptiPRO SFM (Gibco, 12309-019) serum-free cell culture medium, incubated for 1h at 37C, and added to Vero E6 cells (ATCC, CRL-1586). The cells were seeded in 24-well plates at a density of 1.25105 cells/well 24h earlier. Following 1h incubation at 37C, the inoculum was removed and cells were overlaid with a 1:1 mixture of 1.5% (w/v) carboxymethylcellulose in 2xMEM supplemented with 4% FCS. After incubation at 37C for four days, the overlay was removed and the cells were fixed using a 6% formaldehyde solution. Fixed cells were stained with 1% crystal violet solution revealing the formation of plaques. The number of plaques was plotted against the concentration of the surface proteins and a sigmoidal curve was interpolated using GraphPad Prism software version 9.4.1. (681). The top (representing the signal from omicron-not-wild-type-neutralizing antibodies) and bottom (representing the signal from total omicron-neutralizing antibodies) plateaus of each curve were interpolated from a standard curve and divided to obtain the proportion of omicron-not-wild-type-neutralizing antibodies relative to the total omicron-neutralizing antibodies. This fraction was then multiplied with the corresponding quantitative IC50 to obtain the level of omicron-not-wild-type-neutralizing antibodies in plasma.

Cryopreserved PBMC samples were thawed and rested overnight at 37C. The next morning, B cells were isolated immunomagnetically (REAlease CD19 MicroBead Kit, human, Miltenyi Biotec, 130-117-034) following the manufacturers instructions. Briefly, cells were resuspended in the recommended isolation buffer, labeled with anti-CD19 antibodies coupled to magnetic beads, and passed through a magnetic column. B cell-depleted flow-through was collected for the assessment of T cell responses. Immobilized B cells were washed out of the column and enzymatically released from the beads.

To detect the IgG+ B cells specific for the omicron and wild-type SARS-CoV-2 RBD the magnetically isolated B cells were resuspended in FACS buffer (PBS supplemented with 2% FCS, 0.05% NaN3, and 2mM EDTA) and incubated with the fluorescently labeled recombinant RBD proteins (Biotinylated SARS-CoV-2 Spike RBD Protein, Acrobiolabs, SPD-C82E8 and Biotinylated SARS-CoV-2 Spike RBD (B.1.1.529/Omicron), Acrobiolabs, SPD-C82E4). The wild-type RBD protein was conjugated with streptavidin-PE (Biolegend, 405204) and omicron RBD with streptavidin-APC (Biolegend, 40520). 15min into incubation with RBD proteins, an anti-IgG-BV421 antibody (clone G18-145, Biolegend, 562581, diluted 1:20) was added and the incubation was continued for another 15min. Cells were then washed with PBS and stained for viability (ZombieAqua, Biolegend, 423102) for 15min at 4C. Afterward, cells were washed with FACS buffer and incubated with a solution of antibodies blocking human Fc receptors (FcR block, Miltenyi Biotec, 130-059-901, diluted 1:10) for 10min at 4C. Next, a mixture of fluorescently labeled antibodies consisting of: anti-CD3-BV510 (clone UCHT1, Biolegend, 300448, diluted 1:40), anti-CD27-BV605 (clone O323, Biolegend, 302830, diluted 1:20), anti-IgM-BV785 (clone MHM-88, Biolegend, 314544, diluted 1:20), anti-IgA-VioBright 515 (clone REA1014, Miltenyi Biotec, 130-116-886, diluted 1:40), anti-CD21-PE-Cy7 (clone Bu32, Biolegend, 354912, diluted 1:160), and anti-CD19-APC-Cy7 (clone HIB19, Biolegend, 302218, diluted 1:80) was added. Each antibody was checked for performance and titrated before use. Following incubation at 4C for 15min, the cells were washed again and acquired on a BD FACS Celesta flow cytometer with BD FACSDiva Software Version 8.0 (BD Bioscience). Possible longitudinal fluctuations in laser intensity were monitored daily before the experiment using fluorescent beads (Rainbow beads, Biolegend, 422905). The data were analyzed with the FlowJo Software version 10.0.7 (TreeStar). To compensate for the background binding of IgG+ B cells to the fluorescent probes 16 samples were stained with unconjugated streptavidin-PE/APC. The average frequency of streptavidin-PE/APC-binding cells plus two standard deviations was subtracted from the frequencies of RBD-binding cells. No technical replicates were performed due to the scarcity of the samples.

B-cell-depleted PBMC fraction was seeded in 96-well U bottom plates and stimulated with two different pools of overlapping peptides: the first covering the mutated regions of the omicron SARS-CoV-2 spike protein (PepTivator SARS-CoV-2 Prot_S B.1.1.529/BA.1 Mutation Pool, Miltenyi Biotec, 130-129-928) and the second covering conserved regions of the spike (PepTivator SARS-CoV-2 Prot_S B.1.1.529/BA.1 WT Reference Pool, Miltenyi Biotec, 130-129-927). One million cells were stimulated per condition. The final concentration of each peptide was 1g/ml for both peptide pools. Co-stimulatory antibodies (BD FastImmune CD28/CD49d, BD Bioscience, 347690) were added to a final concentration of 1g/ml. For each sample, an equally treated DMSO-stimulated negative control was included. As a positive control, cells were stimulated with PMA (20ng/ml) (Sigma-Aldrich, P1585-1MG) and ionomycin (1g/ml) (Sigma-Aldrich, I3909-1ML). Stimulation was performed at 37C for 6hours. One hour into stimulation Golgi Stop (BD Bioscience, 554724) and Golgi Plug (BD Bioscience, 555029) were added (final concentration 1g/ml) to inhibit vesicular transport and prevent the secretion of the cytokines from cells.

Stimulated cells were washed with PBS and stained for viability (ZombieAqua, Biolegend, 423102) for 15min at 4C. Subsequently, samples were washed with FACS buffer, fixed, and permeabilized in CytoFix/CytoPerm Solution (BD Bioscience, 554714) for 15min at 4C. Fixed cells were washed with 1x Perm/Wash Buffer (BD Bioscience, 554723), and stained for the following intracellular markers; anti-CD3-APC-Cy7 (clone UCHT1, Biolegend, 300426, diluted 1:40), anti-CD4-BV786 (clone SK3, BD Bioscience, 344642, diluted 1:40), anti-CD8-PE-Cy7 (clone SK1, Biolegend, 344712, diluted 1:80), anti-IFN-PE (clone B27, Biolegend, 506507, diluted 1:40), and anti-TNF-BV421 (clone Mab11, Biolegend, 502932, diluted 1:80). Each antibody was checked for performance and titrated before use. Following 15min incubation at 4C, cells were washed thrice with PBS and acquired on a BD FACS Celesta with BD FACSDiva Software Version 8.0 (BD Bioscience). To minimize the signal from unspecific staining only T cells expressing IFN and TNF were considered antigen-specific. The frequencies of antigen-specific T cells were calculated as negative-control-subtracted data. Possible longitudinal fluctuations in laser intensity were monitored daily before the experiment using fluorescent beads (Rainbow beads, Biolegend, 422905). If needed PMT voltages were adjusted to ensure constant signal intensity over time. The data were analyzed with the FlowJo Software version 10.0.7 (TreeStar). No technical replicates were performed due to the scarcity of the samples.

Statistical analysis was performed using GraphPad Prism software version 9.4.1. (681). Differences between the groups were assessed using the Kruskal-Wallis test with Dunns correction for multiple testing. All tests were performed two-sided. Statistical significance is indicated by the following annotations: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

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Vaccination impairs de novo immune response to omicron breakthrough infection, a precondition for the original ... - Nature.com