key: cord-0298607-y98ik7a4 authors: Lin, Wei-Shuo; Chen, I-Chen; Chen, Hui-Chen; Lee, Yi-Chien; Wu, Suh-Chin title: Glycan-masking spike antigen in NTD and RBD elicits broadly neutralizing antibodies against SARS-CoV-2 variants date: 2021-11-02 journal: bioRxiv DOI: 10.1101/2021.11.01.466834 sha: 7bb74d2b1cc7d1d8ea8b1ca2c6abbf446c333cf3 doc_id: 298607 cord_uid: y98ik7a4 Glycan-masking the vaccine antigen by mutating the undesired antigenic sites with an additional N-linked glycosylation motif can refocus B-cell responses to desired/undesired epitopes, without affecting the antigen’s overall-folded structure. This study examine the impact of glycan-masking mutants of the N-terminal domain (NTD) and receptor-binding domain (RBD) of SARS-CoV-2, and found that the antigenic design of the S protein increases the neutralizing antibody titers against the Wuhan-Hu-1 ancestral strain and the recently emerged SARS-CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Our results demonstrated that the use of glycan-masking Ad-S-R158N/Y160T in the NTD elicited a 2.8-fold, 6.5-fold, and 4.6-fold increase in the IC-50 NT titer against the Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2) variants, respectively. Glycan-masking of Ad-S-D428N in the RBD resulted in a 3.0-fold and 2.0-fold increase in the IC50 neutralization titer against the Alpha (B.1.1.7) and Beta (B.1.351) variants, respectively. The use of glycan-masking in Ad-S-R158N/Y160T and Ad-S-D428N antigen design may help develop universal COVID-19 vaccines against current and future emerging SARS-CoV-2 variants. The human codon-optimized S gene of SARS-CoV2 (Wuhan-Hu-1 isolate, accession number MN908947.3) was obtained from GenScript. Site-directed mutagenesis was used to produce the glycan-masking S mutant genes, with the addition of an N-linked glycosylation motif at the S protein residues 135N/N137T, R158N/Y160T, N354/K356T, N370/A372T, G413N, D428N and H519N/P521T. Wildtype S and glycan-masking S genes were first cloned into the pENTR1A vector (Invitrogen), and then cloned into the adenoviral plasmid pAd/CMV/V5-DEST (Invitrogen) using LR ClonaseTM II Enzyme Mix (Invitrogen) to produce the Ad plasmid expressing SARS-CoV-2 S gene. To obtain Ad particles, the Ad plasmids were cleaved with Pac I restriction enzyme to expose the inverted terminal repeats and then transfected into 293A cells separately using TurboFect transfection reagent (Fermentas). After 10-15 d, once the cytopathic effects were visible, the transfected cells and culture media were collected. The cells were disrupted by means of three freeze-thaw cycles to release the intracellular viral particles, and the supernatants of the cell lysates were 4 collected by centrifugation (3000 rpm, 15 min, 4 o C) to obtain the Ad vectors expressing the SARS-Co-V-2 S proteins. To prepare higher titers, the virus was concentrated using a 30-kDa Amicon Ultra-15 Centrifugal Filter (Millipore). The viral stocks were stored at -80 o C. To determine the Ad titers, HEK293A cells were seeded into 6-well plates at a density of 10 6 cells/well and incubated at 37 o C overnight. The 10-fold serially diluted Ad stocks were then added to each well at 37 o C for 24 h. Next, the media containing the diluted Ad vectors were removed, and 3 mL/well of DMEM containing 0.4% agarose and 100 U/ml P/S was added to the 6-well plates. The plaques were visibly quantified 7-10 d after the cells were infected with Ad vectors, and the pfu count was noted. Enzyme-linked immunosorbent assay (ELISA) 5 To measure the SARS-CoV-2 specific total IgG titer in the antisera, recombinant S (Wuhan-Hu-1, catalog number 40589-V08H4), RBD (Wuhan-Hu-1, cat number 40592-V08H), S1 (B. To produce SARS-CoV-2 pseudoviruses, a plasmid expressing the full-length S protein (Wuhan-Hu-1, B.1.1.7, or B.1.351) of SARS-CoV-2 was co-transfected into HEK293T cells with packaging and reporter plasmids pCMVΔ8.91 and pLAS2w.FLuc.Ppuro (RNAi Core, Academia Sinica), using TransIT-LT1 transfection reagent (Mirus Bio). The medium was harvested and concentrated at 48 h posttransfection, followed by estimation of the pseudovirus titer in terms of the luciferase activity of SARS-CoV2-Spp transduction. Serum samples were serially diluted and incubated with 1,000 TU of SARS-CoV-2-pseudotyped lentivirus in DMEM (supplemented with 1% FBS and 100 U/mL P/S) for 1 h at 37°C. The mixture was then inoculated with an equal volume of 10,000 HEK-293T cells stably expressing the ACE2 gene in 96-well plates, which were seeded one day before infection. The culture medium was replaced with fresh complete DMEM (supplemented with 10% FBS, 100 U/mL P/S) at 16 h post-infection and the cells were then continuously cultured for another 48 h before being subjected to a luciferase assay (Promega Bright-GloTM 6 Luciferase Assay System). The percentage of inhibition was calculated as the ratio of the loss of luciferase readout (RLU) in the presence of serum to that of the no serum control. The formula used for the calculation was (RLU Control -RLU Serum) / RLU Control. Neutralization titers (IC50) were measured as the reciprocal of the serum dilution required to obtain a 50% reduction in RLU compared to a control containing the SARS-CoV-2 S-pseudotyped lentivirus only. Neutralization curves and IC50 values were analyzed using the GraphPad Prism 5 Software. Statistical The S protein of SARS-CoV-2 is trimeric, and each monomer comprises of S1 and S2 subunits (30-32). The S1 subunit contains NTD and RBD. To design glycanmasking S antigen(s) for immunization, we used an Ad vector encoding the full-length S gene of the SARS-CoV-2 Wuhan-Hu isolate, by introducing a series of N-linked glycosylation motifs into the S1 region of the S protein, to refocus the antibody responses to the RBD (Fig. 1A) . The sites of glycan-masking were introduced not only in the RBD, but also in the NTD, as RBD and NTD may spatially interact with each other in the quaternary structure of the intact trimeric S protein (Fig. 1B ). The exposed loops or the protruding sites of the exposed loops on the NTD and RBD of the 3-D S 1B) . To characterize the glycan-masking mutations on the S protein, the lysates of HEK293A cells infected with each Ad-S vector were analyzed using 8% SDS-PAGE gels, followed by western blotting with an S1-specific polyclonal antibody. The results indicated the presence of S and S1 in the cell lysates of HEK293A cells infected with variant. To compare these results, the neutralizing IC50 titers from the two separate sets of immunization experiments were normalized to the titer elicited by the wild-type Ad-S against the Wuhan-Hu-1 ancestral strain from. In the first set of immunization experiments, the neutralization IC50 titers elicited in the glycan-masking Ad-S- RBD. However, we were unable to demonstrate the addition of a single N-glycan on the target sites for these glycan-masking Ad-S mutants using SDS-PAGE gel without and with PNGase treatment in western blots, to show an increase in the molecular weights of the glycan-masking mutants, as compared to that of the wild-type Ad-S. It is also likely that glycan-masking mutations may also affect the S protein stabilization for cell surface expression, S/S1 cleavage, and surface S expression. Thus, there is a need for further characterization of these glycan-masking mutants, particularly Ad-S-R158N/Y160T and Ad-S-G413N expressed S proteins. Our results showed that the glycan-masking Ad-S-R158N/Y160T at the N3 loop in the NTD and the glycan-masking Ad-S-N354/K356T and Ad-S-G413N at the C-3 and C-7 loops in the RBD (Fig. S1 ) elicited a potent neutralizing antibody response against the Wuhan-Hu-1 ancestral strain (Fig. 3) . Selection of these glycan-masking sites in this investigation was based on visual inspection of the 3-D S protein structure (PDB ID: 7C2L) for the exposed loops or the protruding sites of the exposed loops in NTD and RBD of the S1 subunit (Fig. S1) . The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. and National Tsing Hua University, Taiwan (109R2807E1, 110Q2805E1). The authors declare no conflict of interest. S A d -S A d -S -F 1 3 5 N / N 1 3 7 T A d -S -R 1 5 8 N / Y 1 6 0 T A d -S -N 3 7 0 / A 3 7 2 T A d -S -H 5 1 9 N / P 5 2 1 T 2 3 RNA virus fitness ViralZone SARS-CoV-2 circulating variants Immunity to SARS-CoV-2 variants of concern Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study Nterminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2 SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants Antibody Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies Neutralizing Activity of BNT162b2-Elicited Serum We thank the RNAi core facility at Academia Sinica for performing the SARS-CoV-2 S-pseudotyped neutralization assay.