Persistent immune imprinting after XBB.1.5 COVID vaccination in humans

Immune imprinting - also known as ‘original antigenic sin’ - describes how the first exposure to a virus shapes the immunological outcome of subsequent exposures to antigenically related strains. SARS-CoV-2 Omicron breakthrough infections and bivalent COVID-19 vaccination were shown to primarily recall cross-reactive memory B cells and antibodies induced by prior mRNA vaccination with the Wuhan-Hu-1 spike rather than priming naive B cells that recognize Omicron-specific epitopes. These findings underscored a strong immune imprinting resulting from repeated Wuhan-Hu-1 spike exposures. To understand if immune imprinting can be overcome, we investigated memory and plasma antibody responses after administration of the updated XBB.1.5 COVID mRNA vaccine booster. Our data show that the XBB.1.5 booster elicits neutralizing antibody responses against current variants that are dominated by recall of pre-existing memory B cells previously induced by the Wuhan-Hu-1 spike. These results indicate that immune imprinting persists even after multiple exposures to Omicron spikes through vaccination and infection, including post XBB.1.5 spike booster mRNA vaccination, which will need to be considered to guide the design of future vaccine boosters.

Experiments 1 and 2 (exp. 1 and exp.2) were performed using a first batch of VSV S pseudotyped viruses using VeroE6-TMPRSS2 cells puromycin resistant.Experiments 3 and 4 (exp.3 and exp 4) were performed using a second batch of VSV S pseudotyped viruses using VeroE6-TMPRSS2 cells geneticin resistant for exp. 3 and VERO-TMPRSS2 cell puromycin resistant for exp. 4.
Table S2.Demographics information of the subjects in the Wuhan-Hu-1/BA.5 bivalent vaccinee cohorts

Cell lines
Cell lines used in this study were obtained from JCRB-Cell Bank (VeroE6-TMPRSS2, geneticin resistant) and ThermoFisher Scientific (Expi293F™ cells).Cells were cultivated at 37°C, in an atmosphere of 5 % CO 2 and with 130 rpm of agitation for suspension cells.None of the cell lines used were routinely tested for mycoplasma contamination.The VeroE6-TMPRSS2 puromycin resistant cells were previously described 22 .
Recombinant protein production SARS-CoV-2 Wuhan-Hu-1 and XBB.1.5HexaPro S ectodomains were expressed in Expi293 cells at 37°C and 8% CO 2 .Cells were transfected with the corresponding plasmids using Expifectamine following the manufacturer's instructions.Four days post-transfection, supernatants were clarified by centrifugation at 4121g for 30 minutes, supplemented with 25 mM phosphate pH 8.0, 300 mM NaCl, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF).Supernatant was then 0.22µm vacuum filtered and passed through 1 mL His trap HP or Excel column (Cytiva) previously equilibrated in 25 mM phosphate pH 8.0, 300 mM NaCl. S proteins were eluted using a buffer identical to the binding buffer with the addition of 300 mM imidazole.
Fractions containing the proteins were pooled and buffer exchanged to 50 mM Tris-HCl pH 8.0, 150 mM NaCl and stored at 4°C or immediately used.
The SARS-CoV-2 Wu and XBB.1.5RBD proteins were expressed and purified as described above.Following buffer exchange, the purified RBDs were biotinylated using the BirA biotinprotein ligase reaction kit (Avidity).The biotinylated proteins were passed, washed, and eluted again on the same affinity column, concentrated and ran over a Superdex200 increase 10/300 size-exclusion column (Cytiva).Fractions corresponding to monomeric and monodisperse RBDs were collected, flash frozen, and stored at -80°C until use.

Plasma antibody depletion
Invitrogen His-Tag Dynabeads (ThermoFisher 10104D) were used for depletion of plasma samples from antibodies recognizing the Wuhan-Hu-1 S trimer, as previously described 9 with some modifications.Vortexed beads were incubated at room temperature on an Invitrogen DynaMag-2 Magnet (ThermoFisher 12-321-D) for two minutes to allow beads to separate for the liquid phase.Supernatant was discarded and beads were washed one time with TBS-T and divided in two tubes.After a two-minute incubation on the magnet, TBS-T supernatants were discarded and one set of beads was incubated with 4 mg of purified his-tagged SARS-CoV-2 Wuhan-Hu-1 S ectodomain trimer (Wuhan-Hu-1 S depletion) and the other set was incubated with TBS-T alone (mock depletion) with gentle rotation for 1 h at room temperature.Supernatants were discarded using the magnet and beads were washed three times with TBS-T.Subsequently, 20 µl of each of the plasma samples were incubated with 80 µl of S-loaded beads or mock-loaded beads for 1 h at 37°C during which time they were mixed every 15 min.Plasma samples were recovered using the magnet to separate the beads and used for neutralization assays.

VSV pseudotyped virus production
Vesicular stomatitis virus (VSV) were pseudotyped with the SARS-CoV-2 S proteins corresponding to Wuhan-D614G, BQ.1.1,XBB.1.5 and BA.2.86 variants following a previously described protocol 26 .Briefly, HEK293T cells seeded in poly-D-lysine coated 10-cm dishes in DMEM supplemented with 10% FBS and 1% PenStrep were transfected with a mixture of 24 µg of the corresponding plasmids, 60 µl Lipofectamine 2000 (Life Technologies) in 5 ml of Opti-MEM, following the manufacturer's instructions.After 5 h at 37°C, 5 ml of DMEM supplemented with 20% FBS and 2% PenStrep were added.The next day, cells were washed three times with DMEM and were transduced with VSVΔG-luc 27 .After 2 h, the virus inoculum was removed and cells were washed five times with DMEM prior to the addition of DMEM supplemented with anti-VSV-G antibody [Il-mouse hybridoma supernatant diluted 1 to 25 (v/v), from CRL-2700, ATCC] to minimize parental background.After 18-24 h, supernatants containing pseudotyped VSV were harvested, centrifuged at 2,000 x g for 5 min to remove cellular debris, filtered with a 0.45 µm membrane, concentrated 10 times using a 30 kDa cut off membrane (Amicon), aliquoted, and frozen at -80°C.

Pseudotyped VSV neutralization
VeroE6-TMPRSS2 cells were seeded into coated clear bottom white walled 96-well plates at 40,000 cells/well and cultured overnight at 37°C.Eleven 3-fold serial dilutions of each plasma sample were prepared in DMEM.Pseudotyped VSV viruses, diluted 1 to 20 in DMEM containing anti-VSV-G antibody, were added 1:1 (v/v) to each plasma sample dilution and mixtures of 50 µl volume were incubated for 45-60 min at 37°C.VeroE6-TMPRSS2 cells were washed three times with DMEM and 40 μL of the mixture containing pseudotyped virus and plasma samples were added.Two hours later, 40 μL of DMEM were added to the cells.After 17-20 h, 70 μL of One-Glo-EX substrate (Promega) were added to each well and incubated on a plate shaker in the dark at 37°C.After 5-15 min incubation, plates were read on a Biotek Neo2 plate reader.Measurements were done in duplicate or triplicate with at least two biological replicates.Relative luciferase units were plotted and normalized in Prism (GraphPad): cells without pseudotyped virus added were defined as 0 % infection or 100 % neutralization, and cells with virus only (no plasma) were defined as 100 % infection or 0 % neutralization.

Enzyme-linked immunosorbent assays (ELISA)
Analysis of plasma binding antibodies for samples mock-depleted or depleted of antibodies binding to the Wuhan-Hu-1 S ectodomain trimer was performed using ELISAs.Briefly, clear flat bottom Immuno Nonsterile 384-well plates (Thermo Scientific) were coated overnight at room temperature with 30 μl of Wuhan-Hu-1 S or XBB.1.5S prepared at 3 μg/ml in PBS (137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, and 1.8 mM of KH2PO4, pH 7.2).The next day, plates were blocked with Blocker™ Casein (Thermo Scientific) and subsequently incubated with serial dilutions of plasma samples for 1 h at 37°C.After four washing steps with TBS-T, goat anti-human IgG-Fc secondary antibody conjugated to HRP (Invitrogen A18817, diluted 1/500) was added and incubated for 1 h at 37°C.Plates were washed four times with TBS-T and KPL SureBlue Reserve™ TMB Microwell Peroxidase Substrate (VWR) was added.After 2 min incubation, 1N HCl was added and absorbance at 405 nm was measured using a Biotek Neo2 plate reader.Data were plotted using GraphPad Prism 9.1.0.

Flow cytometry analysis of SARS-CoV-2 RBD-reactive memory B cells
To define specific B cell populations reactive with the XBB.1.5 and the Wuhan-Hu-1 RBDs, RBD-streptavidin tetramers conjugated to fluorophores were generated by incubating biotinylated RBDs with streptavidin at a 4:1 molar ratio for 30 min at 4 °C.Excess of free biotin was then added to the reaction to bind any unconjugated sites in the streptavidin tetramers.The RBD-streptavidin tetramers were washed once with PBS and concentrated with a 100-kDa cutoff centrifugal concentrator (Amicon).An additional streptavidin tetramer conjugated to biotin only was generated and included in the staining as decoy.
Approximately 5 to 15 million PMBCs were collected 7 to 13 days post-vaccination for individuals who received an XBB. ), along with the RBD-streptavidin tetramers for 30 min at 4°C.Cells were washed three times, resuspended in PBS, and passed through a 35-μm filter before being examined on a BD FACSymphony A3 for acquisition and FlowJo 10.8.1 for analysis.Gates for identifying the XBB.1.5RBD double-positive population as well as the subsequent Wuhan-Hu-1 RBD-positive and Wuhan-Hu-1 RBD-negative populations were drawn based on staining of fluorescent minus one controls.

Figure S1 .
Figure S1.Plasma neutralizing antibody titers after vaccination with the XBB.1.5S mRNA booster (A) and after the bivalent Wuhan-Hu-1/BA.5 S mRNA booster (B).Dose-response curves for all repeats are shown with each patient ID indicated on top of the graphs.Experiments 1 and 2 (exp. 1 and exp.2) were performed using a first batch of VSV S pseudotyped viruses using VeroE6-TMPRSS2 cells puromycin resistant.Experiments 3 and 4 (exp.3 and exp 4) were performed using a second batch of VSV S pseudotyped viruses using VeroE6-TMPRSS2 cells geneticin resistant for exp. 3 and VERO-TMPRSS2 cell puromycin resistant for exp. 4.

Figure S2 .
Figure S2.Plasma antibody binding titers against Wuhan-Hu-1 S and XBB.1.5S after vaccination with the XBB.1.5S mRNA booster in plasma samples mock-depleted and depleted of Wuhan-Hu-1 S-directed antibodies.Dose-response curves for one representative experiment are shown.The color key indicates patient IDs.

Figure S3 .
Figure S3.Plasma neutralizing antibody titers after vaccination with the XBB.1.5S mRNA booster in plasma samples mock-depleted and depleted of Wuhan-Hu-1 S-directed antibodies.Dose-response curves for one representative experiment are shown.Patient IDs are indicated on top of each graph.

Figure S4 .
Figure S4.Flow cytometry analysis of memory B cells.Panel A shows the gating strategy to evaluate the cross-reactivity with the Wuhan-Hu-1 RBD of XBB.1.5RBD+ memory B cells.Dump includes markers for CD3, CD8, CD14, and CD16.Panel B shows the gating of XBB.1.5RBD-reactive memory B cells and subsequent Wuhan-Hu-1 RBD-binding of these memory B cells for each individual profiled.