Neutralization assays were performed 13 times in at least triplicates for each dilution. as the most dominant strain worldwide. With 37 amino acid substitutions in the Spike protein (S), it marks a major antigenic shift from the original Wuhan-Hu1 and the delta sequence. Immune evasion is a major contributing factor foromicrons improved transmissibility13. To date, all approved vaccines against SARS-CoV-2 are based on the Wuhan-Hu1 sequence of the S protein. Senktide Serologic studies have shown that convalescent patient plasma and plasma from individuals vaccinated with BNT162b2 neutralize omicron 44-fold and 12-fold less effectively than delta2. A third immunization with BNT162b2 (booster) increases neutralization efficacy by 10 to 100-fold2,4. Accordingly, the booster prevents 88% of omicron-related hospitalizations, as opposed to only 52% at 25 + weeks post second vaccination5,6. This increased efficacy should be attributed to higher anti-S antibodies with increased affinity to Wuhan-Hu1 and to omicron only by proxy. While sera and plasma neutralization levels have been established, the anti-omicron response to BNT162b2 is not well understood on a single antibody level. We recently investigated the acute B cell response to the BNT162b2 vaccine on a single-cell level and discerned the development of anti-receptor binding domain (RBD) and anti-S2 antibodies from nave and memory B cells, respectively7. The RBD is located on the S1 domain of the S protein and Rabbit polyclonal to KLF8 interacts with human angiotensin-converting enzyme 2 (ACE2) to initiate viral cell entry, whereas the S2 domain facilitates viral cell membrane fusion8,9. Most neutralizing antibodies found in COVID-19 patients target RBD10. Neutralizing antibodies against the S2 subunit have been described, but they generally neutralize SARS-CoV-2 less efficiently than anti-RBD mAbs11,12. However, we have shown that anti-S2 mAbs develop early in response to vaccination, and are cross-reactive to other betacoronaviruses due to higher structural conservation of the S2 subunit over S17,13,14. Anti-S2 antibodies could therefore be crucial for the protection against novel variants. We previously expressed and tested 50 vaccine-derived monoclonal antibodies (mAbs) against RBD/S1 and S2, and identified 15 anti-RBD mAbs that neutralized Wuhan-Hu1 and delta7. Here, we added additional 55 mAbs from the same sequence dataset and investigated their binding to RBD and S2 as well as their neutralization potency to Wuhan-Hu1, delta, and omicron. == Results == Of the 105 included mAbs, a total of 35 bound to RBD (Fig. 1a), of which 23 neutralized Wuhan-Hu1 pseudovirus with variable potency (Fig. 1b,c). 16 of 23 Wuhan-Hu1-neutralizing anti-RBD mAbs neutralized delta and 13 neutralized omicron (Fig. 1b). Neutralization potencies for omicron were decreased 19-fold compared to Wuhan-Hu1 (median half-maximum inhibitory concentration (IC50) Wuhan-Hu1:1.28 nM, omicron: 24.33 nM) (Fig. 1c). The best neutralizing mAb against omicron was 46-fold less potent than the best neutralizing mAb against Wuhan-Hu1 (min. IC50 Wuhan-Hu1: 86 pM, omicron: 4.01 nM). In contrast, overall neutralization potency to delta was only 4.6-fold decreased (median IC50 Wuhan-Hu1: 1.28nM, delta: 5.93 nM) and maximum neutralization potencies were in a similar range for Wuhan-Hu1 and delta (min. IC50 Wuhan-Hu1: 86 pM, delta: 24 pM) (Fig. 1c). == Figure 1. Binding and neutralization of BNT162b2-derived anti-RBD antibodies. == (a)ELISA measurements of anti-RBD mAbs to Wuhan-Hu1 RBD, mean AUC SD.(b)Neutralization (IC50) of individual anti-RBD mAbs against Wuhan-Hu1, delta, and Senktide omicron pseudovirus. Arrows indicate Senktide non-neutralization of the respective antibody against the variant of the same color, dashed line at IC50 = 10nM facilitates comparison with anti-S2 antibodies inFig. 2b.(c)Comparison of IC50 values of anti-RBD mAbs against Wuhan-Hu1, delta, and omicron. Medians interquartile ranges are shown; ns, not significant; ***P = 0.0002, unpaired two-tailed Kruskal-Wallis test, Dunn-corrected Senktide for multiple comparisons. In addition, we identified 12 anti-S2 mAbs, of which 7 neutralized Wuhan-Hu1 pseudovirus (Fig. 2a,b). As previously described, most vaccine-derived anti-S2 antibodies cross-reacted with the betacoronaviruses OC43 and HKU1 (Fig. 1a)7. The anti-S2 mAbs neutralize Wuhan-Hu1 with 16.9-fold lower potency than anti-RBD.