Broadly neutralizing antibodies target the coronavirus fusion peptide

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We use an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

Binding of secreted antibody to the beads was detected in the CY5 or TRED channels by capturing images at 6 min intervals over a 30 min time course. In the second step of the assay, the MERS/OC43 beads were replaced with 7 μm streptavidin beads coated with 10 μg/mL SARS-CoV-2 spike, and antibody binding was detected as before. OEP light cages were applied to export individual cross-reactive monoclonal antibody (mAb)-secreting B cells directly into Dynabeads mRNA DIRECT lysis buffer (Life Technologies, 61011) in 96-well plates. Plates were sealed with Microseal foil film (BioRad, MSF1001) and immediately frozen on dry ice before transferring to -80 °C for long-term storage. mAb sequence analysis and expression Heavy and light chain sequences were amplified from single B cell lysates using RT-PCR (20,43,52) and resolved by Sanger Sequencing (Eurofins and Genewiz). Analyses of the heavy chain variable (VH) and (lambda/kappa variable) Vλ/Vκ genes, complementarity-determining region 3 (CDR3) sequences, and percentage of somatic mutations were carried out using Geneious Prime  Human leukocyte antigen (HLA) typing for donor identification Several mAbs were isolated from screens that involved pooling of B cells from two different individuals. To identify the source donor of these mAbs, a commercially available ScisGo ® -HLA-v6 kit (Scisco Genetics Inc., Seattle WA) employing an amplicon-based sequencing by synthesis approach was used to determine HLA types of amplified complementary DNA (cDNA) from single cell isolates. The approach uses a two-stage amplicon-based PCR for locus amplification and sample barcoding. Although designed for amplification from genomic DNA, a subset of the kit amplicons was functional in amplifying product from cDNA. Briefly, samples were sequentially applied to stage 1 (S1) and stage 2 (S2) PCR amplification according to the manufacturer supplied protocol. After amplification, the reactions were combined, purified, and applied to a MiSeq using Illumina Version 2 chemistry with 500-cycle, paired-end sequencing (Illumina, San Diego, CA). Data assembly and analysis were performed using Sciscloud ® (Scisco Genetics Inc., Seattle WA) computational tools adapted specifically to assemble HLA genomic sequences derivative from the ScisGo ® -HLA-v6 kit. Amplified portions of the HLA class I and class II genes were compared with prior typing data allowing the unambiguous identification of corresponding samples. Access to all software for data transfer and analysis was included as a component of the kit and made available through a web browser.

Recombinant mAb binding to coronavirus antigens
Recombinant mAbs were diluted 4-fold in 0.05% BSA w/v in PBS to generate a 47.7 ng/mL -200 μg/mL dilution series. Multiplexed antigen-labelled beads were incubated with mAb titrations for 30 min at room temperature, then washed and stained with 2.5 μg/mL goat anti-human IgG Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-170). Samples were acquired on the iQue Screener Plus and data were analysed with FlowJo. Data points from the titration curves were interconnected without logistic regression and AUC analyses were performed with GraphPad Prism and reported after correction using the AUC of the negative control CD4 population. ThCoV-HKU12 (Uniprot accession # B6VDX8), TCoV (Uniprot accession # B3FHU5), WiCoV-HKU20 (Uniprot accession # H9BR25) were aligned using MAFFT v7 server using a BLOSUM62 scoring matrix and L-INS-i algorithm. The sequence alignment was used to generate a sequence logo plot using the Weblogo 3.0 server (56)and to color conserved amino acid residues on a prefusion stabilized spike protein (PDB 6VSB) using Chimera X.
SARS-CoV-2 spike and S2 subunit epitope binning by surface plasmon resonance (SPR) Epitope binning experiments were performed on the Carterra LSA with cross-reactive mAbs coupled to an HC30M chip (Carterra).

Imaging-based fusion inhibition assay
HeLa cell lines (Expasy CVCL_0030) stably expressing either CoV spike proteins or their cognate receptor were generated as previously reported (15)  Hoechst for 10 min and washed twice with PBS. Images were acquired in A488, A568 and DAPI channels using a BZ-X fluorescence microscope (KEYENCE) and processed using Fiji ImageJ (57).

Colorimetric fusion inhibition assay
HEKBlue cells (Invivogen, hkb-hace2tpsa) stably transfected to express human angiotensin-I- hours. Absorbance was measured using an EnSpire Multimode (Perkin Elmer) plate reader at 635 nm. Percent inhibition was calculated as described in (58), briefly (1-(E-N)/(P-N)) x 100; where "E" is absorbance of the antibody treatment group, "N" is the absorbance of vector control, and "P" is the absorbance of the no antibody treatment group.
Shotgun mutagenesis epitope mapping of antibodies by alanine scanning Epitope mapping was performed essentially as previously described (59), using a SARS-CoV-2 (Wuhan Hu-1 strain) S2 subunit shotgun mutagenesis mutation library, made using a full-length expression construct for the SARS-CoV-2 spike glycoprotein. 513 S2 residues (between residues 689 -1247) were mutated individually to alanine, and alanine residues to serine. Mutations were confirmed by DNA sequencing, and clones arrayed in a 384-well plate, one mutant per well.
Binding of mAbs to each mutant clone in the alanine scanning library was determined, in duplicate, by high-throughput flow cytometry. A plasmid encoding cDNA for each spike protein mutant was transfected into HEK-293T cells and allowed to express for 22 h. Cells were fixed in 4% (v/v) PFA (Electron Microscopy Sciences), and permeabilized with 0.1% (w/v) saponin (Sigma-Aldrich) in PBS before incubation with mAbs diluted in PBS, 10% normal goat serum (Sigma), and 0.1% saponin. mAb screening concentrations were determined using an independent immunofluorescence titration curve against cells expressing wild-type spike protein to ensure that signals were within the linear range of detection. Antibodies were detected using 3.75 μg/mL of Alexa-Fluor-488-conjugated secondary antibodies (Jackson ImmunoResearch) in 10% normal goat serum with 0.1% saponin. Cells were washed three times with PBS/0.1% saponin followed by two washes in PBS, and mean cellular fluorescence was detected using a high-throughput Intellicyt iQue flow cytometer (Sartorius). Antibody reactivity against each mutant spike protein clone was calculated relative to wild-type spike protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type spike-transfected controls.
Mutations within clones were identified as critical to the mAb epitope if they did not support reactivity of the test mAb but supported reactivity of other SARS-CoV-2 antibodies. This counterscreen strategy facilitates the exclusion of spike protein mutants that are locally misfolded or have an expression defect.
Where NC is the average readout of negative controls (wells without live virus), and PC is the Core and Pathology data packages were received. Animals were randomly assigned to groups to balance as closely as possible between ages (same in this experiment), weight ranges, and sex distribution prior to challenge in consultation with a NIH statistician.