Broad anti-SARS-CoV-2 antibody immunity induced by heterologous ChAdOx1/mRNA-1273 vaccination

Heterologous prime-boost immunization strategies have the potential to augment COVID-19 vaccine efficacy. We longitudinally profiled SARS-CoV-2 spike (S)-specific serological and memory B cell (MBC) responses in individuals receiving either homologous (ChAdOx1:ChAdOx1) or heterologous (ChAdOx1:mRNA-1273) prime-boost vaccination. Heterologous mRNA booster immunization induced higher serum neutralizing antibody and MBC responses against SARS-CoV-2 variants of concern (VOCs) compared to homologous ChAdOx1 boosting. Specificity mapping of circulating B cells revealed that mRNA-1273 boost immunofocused ChAdOx1-primed responses onto epitopes expressed on prefusion-stabilized S. Monoclonal antibodies isolated from mRNA-1273 boosted participants displayed overall higher binding affinities and increased breadth of reactivity against VOCs relative to those isolated from ChAdOx1-boosted individuals. Overall, the results provide molecular insight into the enhanced quality of the B cell response induced following heterologous mRNA booster vaccination.


Recombinant antigen production.
For production of prefusion-stabilized SARS-CoV-2 S-2P, DNA encoding residues 1-1208 of the SARS-CoV-2 spike with "PP" mutations at positions 986 and 987, "GSAS" mutations from positions 682-685 and a C-terminal T4 fibritin motif, 8X HisTag and TwinStrepTag (SARS-CoV-2 S-2P) was cloned into a pcDNA3.4 vector and transiently transfected into FreeStyle HEK 293F cells (Thermo Fisher) using polyethylenimine. Following one week of culture, the supernatants were harvested, centrifuged to remove cellular debris, and purified by Ni affinity chromatography. Recombinant protein was further purified by size exclusion chromatography using the Superose 6 column (GE Healthcare). were pre-incubated with serial dilutions of heat-inactivated sera for one h at 37 ˚C and then added to a confluent monolayer of Vero-E6 cells. After one h of incubation, cell culture supernatants were aspirated and exchanged with fresh infection medium. Cells were subsequently incubated for 16-24 hrs at 37 ˚C with 5% carbon dioxide. Next, cells were washed with DPBS, fixed with 10% formalin for 15 min at room temperature, and permeabilized with 70% ethanol. Viral infection was detected by VirSpot immunostaining, with primary detection using an anti-nucleocapsid protein antibody followed by secondary detection with peroxidaseconjugated anti-human IgG and KPL TrueBlue substrate. Blue precipitates, which indicate the presence of viral N protein, were quantified by a CTL ImmunoSpot analyzer to determine the 50% serum neutralizing titers, as described previously (30).
Authentic SARS-CoV-2 CPE microneutralization assay against Delta. activity. Following incubation, extra neutral red stain was washed away, and remaining neutral red that was retained by cellular lysosomes was extracted using a solution of 50% ethanol and 1% acetic acid. The amount of neutral red was quantified by absorbance at 450 nm using a spectrophotometer. To calculate percentage neutralization, the average absorbance of cells infected in the presence of serum dilutions was subtracted by the absorbance of cells infected in the absence of serum and then normalized to cells that were not infected with virus. To calculate the 50% neutralization titer, neutralization curves were fit by non-linear regression using a fourparameter sigmoidal dose-response model in GraphPad Prism (version 9).

Authentic SARS-CoV-2 CPE microneutralization assay against Omicron.
Microneutralization based on cytopathic effects (CPE) was performed essentially as previously described (31). Briefly, serum was 3-fold serially diluted, mixed with virus, incubated for 1 hour and finally added, in duplicates, to confluent Vero E6 cells in 96-well plates. An authentic Omicron isolate was obtained from a Swedish patient. After 5 days of incubation, wells were inspected for signs of CPE by optical microscopy. Each well was scored as either neutralizing (if no signs of CPE was observed) or non-neutralizing (if any CPE was observed).
The arithmetic mean neutralization titer of the reciprocals of the highest neutralizing dilutions from the two duplicates for each sample was then calculated to determine the IC100.

SARS-CoV-2 MLV pseudovirus neutralization assay.
Neutralizing activity of monoclonal antibodies was evaluated in an MLV-based pseudovirus assay. HeLa-hACE2 reporter cells (BPS Bioscience Cat #79958) were seeded overnight at 10,000 to 15,000 cells per well in 96-well tissue culture plates (Corning). For screening of antibody neutralization activity at a single concentration, 25 µl of SARS-CoV-2 Spseudotyped MLV particles were added to IgG in MEM/EBSS supplemented with 10% FBS to achieve a final antibody concentration of 1 µg/ml in a 100 µl volume. For determination of 50% inhibitory concentrations, MLV particles were mixed with serial dilutions of antibodies ranging from 10 µg/ml to 0.02 ng/ml. The mixture of viral particles and antibodies was incubated for 1 h at 37 ˚C with 5% carbon dioxide. Separately, HeLa-hACE2 cells were washed three times with DPBS. The virus-antibody solution was subsequently incubated with HeLa-hACE2 cells for 72 hours, after which cells were lysed with Luciferase Cell Culture Lysis 5× reagent (Promega).

Amplification and cloning of antibody variable genes.
Antibody variable gene transcripts (VH, Vκ, Vλ) were amplified by RT-PCR using SuperScript IV enzyme (ThermoFisher Scientific), followed by two cycles of nested PCRs, as described previously (33). The second round of nested PCR contained 40 base pairs of 5' and 3' homology to restriction enzyme-digested S. cerevisiae expression vectors to enable homologous recombination during transformation. Briefly, amplified variable gene transcripts were chemically transformed into competent yeast cells using the lithium acetate method and plated on selective amino acid drop-out media (34). Individual yeast colonies were subsequently picked for sequencing and characterization.

Expression and purification of IgGs and Fabs.
IgGs were expressed in S. cerevisiae cultures grown in 24-well plates, as described previously (33). Briefly, after 6 days of cell culture growth, the IgG-containing supernatant was harvested by centrifugation. IgGs were purified by protein A-affinity chromatography, eluted with 200 mM acetic acid/50 mM NaCl (pH 3.5), and pH-neutralized with 1/8 th volume of 2 M Hepes (pH 8.0) Fab fragments were generated by digestion of IgG with papain for 2 hrs at 30 °C before terminating the reaction with iodoacetamide. The digested mixture of Fab and Fc was subsequently purified first by Protein A agarose to remove Fc fragments and undigested IgG.

Bio-Layer Interferometry Kinetic Measurements (BLI).
Apparent binding affinities were measured by biolayer interferometry (BLI) using a were then allowed to incubate in PBSF for a minimum of 30 min. After a short (60 s) baseline step in PBSF, the antigen-loaded biosensors were exposed (180 s) to the IgG or Fab (100 nM) and then dipped (180 s) into PBSF to measure any dissociation of the IgGs from the biosensor surface. Data for which binding responses were >0.1 nm were aligned and fitted as described above.

Competitive binding analysis using BLI.
Antibody competition with hACE2 for binding to SARS-CoV-2 RBD and with the anti-S2 antibody ADI-69962 for binding to recombinant SARS-CoV-2 WT S was evaluated by BLI using a ForteBio Octet HTX (Sartorius). All binding steps were performed at 25 o C and at an orbital shaking speed of 1000 rpm. All reagents were formulated in 1X PBS with 0.1% w/v BSA.
For ACE2 competition, IgGs (100 nM) were captured to anti-human IgG capture (AHC) biosensors (Molecular Devices) to a sensor response of 1.0 nm-1.4 nm. IgG-loaded sensors were soaked (20 min) in an irrelevant IgG1 solution (0.5 mg/ml) to block remaining Fc binding sites and were allowed to equilibrate in PBSF for a minimum of 30 min. To assess any cross interactions between proteins on the sensor surface and the secondary molecules, the loaded and blocked sensors were exposed (90 s) to hACE2 receptor (300 nM) prior to the binning analysis.
For ADI-69962 (anti-S2 antibody) competition, ADI-69962 IgG (100 nM) was captured to anti-human IgG biosensors to a sensor response of 1.0 nm-1.4 nm. IgG-loaded sensors were soaked (20 min) in an irrelevant IgG1 solution (0.5 mg/ml) to block remaining Fc binding sites and were allowed to equilibrate in PBSF for a minimum of 30 min. Loaded and blocked sensors were exposed (90 s) to each sample antibody (300 nM) to assess any non-specific cross interactions between the sample IgG and ADI-69962 before exposure (180 s) to SARS-CoV-2 WT S (100 nM). Since WT S exists in a trimeric form, tips were exposed (180 s) to ADI-69962 (100 nM) in solution to block any remaining accessible epitopes present on protomers not directly bound to ADI-69962 loaded on the biosensor. Lastly, ADI-69962 and WT S-loaded tips were exposed (180 s) to sample IgG (100 nM). The data was y-axis normalized, and interstep corrected using the FortéBio Data Analysis Software version 11.0. Additional binding by the secondary molecule indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor). Fig. S1. Serum IgG binding to SARS-CoV-2, HKU1, and OC43 S antigens, as assessed by ELISA. Pre-pandemic samples were included as negative controls to assess baseline reactivities.