performed transfection for S2 and beta-CoV antibody productions; S.W. analyses indicated WS6 to neutralize by inhibiting fusion and post-viral connection. Evaluation of WS6 with various other recently determined antibodies that broadly neutralize beta-coronaviruses indicated a stem-helical supersitecentered on hydrophobic residues Phe1148, Leu1152, Tyr1155, and Phe1156to be considered a promising focus on for vaccine style. Keywords:beta-coronavirus, neutralizing antibody broadly, COVID-19, crystal framework, SARS-CoV-2, S2-aimed antibody, vaccine style == Graphical abstract == Shi et al. determined a wide beta-coronavirus neutralizing antibody from mice immunized with mRNA encoding the SARS-CoV-2 spike. This antibody goals an S2 supersite composed of a hydrophobic cluster spanning three helical transforms, that are conserved among beta-coronaviruses. This S2 supersite is apparently a good focus on for wide beta-coronavirus vaccines. == Launch == The coronavirus disease 2019 (COVID-19) pandemic, caused by the Quinagolide hydrochloride zoonotic infections of severe severe respiratory symptoms coronavirus 2 (SARS-CoV-2), provides lasted more than 2 years with more than 500 million cases and over 6 million deaths (https://www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19). Continuously evolving variants are making current licensed vaccines less effective (Araf et al., 2022;Edara et al., 2021;Garcia-Beltran et al., 2022;Liu FGF18 et al., Quinagolide hydrochloride 2021;Pajon et al., 2022). Vaccines capable of neutralizing all SARS-CoV-2 variants for the foreseeable future are of high interest. Antibodies with broad neutralizing capacity are also of interest: if ultrapotent, they might be useful as therapeutic antibodies, but even if they are only of moderate potency, their epitopes are useful as vaccine templates (Kong et al., 2016). Virtually all SARS-CoV-2 neutralizing antibodies are directed against the trimeric ectodomain of the spike glycoprotein, which comprises two subunits, S1 and S2. Neutralizing antibodies isolated from COVID-19 convalescent donors or from vaccinees after spike immunization are directed primarily against the N-terminal domain (NTD) or receptor-binding domain (RBD) on the S1 subunit of the trimeric viral surface spike glycoprotein (spike) (Barnes et al., 2020;Brouwer et al., 2020;Cao et al., 2020;Cerutti et al., 2021;Ju et al., 2020;Liu et al., 2020;McCallum et al., 2021;Robbiani et al., 2020;Rogers et al., 2020;Seydoux et al., 2020;Suryadevara et al., 2021;Zost et al., 2020). Several recently emerged SARS-CoV-2 variants, such as Delta and Omicron, evade these antibodies by mutations that reduce or knock out antibody binding but maintain or even enhance infectivity (Garcia-Beltran et al., 2022;Liu et al., 2021;Sievers et al., 2022;Syed et al., 2022). Antibodies against most other regions on the spike are generally poorly neutralizing to non-neutralizing; several antibodies, however, such as antibodies 28D9 (Wang et al., 2021a) and S2P6 (Pinto et al., 2021) have been reported to neutralize diverse strains of beta-coronaviruses through recognition of a stem-helix supersite of vulnerability in the S2 subunit (Hsieh et al., 2021;Li et al., 2022;Sauer et al., 2021;Zhou et al., 2021). To investigate the breadth of neutralizing antibodies obtained from mice vaccinated by mRNA encoding the SARS-CoV-2 spike, we assessed monoclonal antibodies for the location of their epitopes, the breadth of their binding Quinagolide hydrochloride to diverse spikes, and their neutralization capacities. We found one antibody, WS6, with broad binding capacity and moderate neutralization potency, and we determined its crystal structure in complex with its epitope, the step in the entry pathway where it neutralized, and how its recognition compared with other recently identified antibodies with overlapping epitopes. The results reveal a highly promising vaccine target in the S2 subunitcomprising a hydrophobic cluster spanning three helical turns, with acidic residues framing the central turnand add WS6 to the panel of antibodies by which to guide its vaccine development. == Results == == Identification and characterization of SARS-CoV-2 spike-specific antibodies from immunized mice Quinagolide hydrochloride == To obtain antibodies specific for SARS-CoV-2 spike glycoprotein, we immunized mice with mRNA coding for SARS-CoV-2 spike (Figure 1A). To generate hybridomas, we boosted with soluble spike protein and, after 3 days, generated hybridomas by fusing splenocyte B cells with Sp2/0 cells from the mouse with the highest plasma neutralization titers to SARS-CoV-2. Eleven monoclonal antibodies, named WS1 to WS11, bound SARS-CoV-2 S-dTM (spike residues 11,206) by ELISA (Figure 1B). Nine of these bound the S1 subunit, either S1-short1 (spike residues 1670) or S1R (residues 1537). Six of them, WS1, WS2, WS3, WS7, WS8, and WS10, bound NTD, and three of them, WS4, WS9, and WS11, bound RBD. Antibodies WS5 and WS6, however, did not bind NTD, RBD, or Quinagolide hydrochloride S1, and their binding epitopes were presumably on the S2 subunit of the spike. == Figure 1. == Spike-mRNA-immunized mice elicit antibodies against diverse regions of spike, several of which bound diverse beta-coronavirus spikes and one of which, WS6, neutralized them (A) Immunization scheme. NatSP is the.