key: cord-0270313-q44n0385 authors: Thébault, Stéphanie; Lejal, Nathalie; Dogliani, Alexis; Donchet, Amélie; Urvoas, Agathe; Valerio-Lepiniec, Marie; Lavie, Muriel; Baronti, Cécile; Touret, Franck; da Costa, Bruno; Bourgon, Clara; Fraysse, Audrey; Saint-Albin-Deliot, Audrey; Morel, Jessica; Klonjkowski, Bernard; de Lamballerie, Xavier; Dubuisson, Jean; Roussel, Alain; Minard, Philippe; Le Poder, Sophie; Meunier, Nicolas; Delmas, Bernard title: Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals date: 2022-05-11 journal: bioRxiv DOI: 10.1101/2022.05.10.491295 sha: 4ac2932ace2ff3287c40fa2714330d5789fd7b9f doc_id: 270313 cord_uid: q44n0385 The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment. Author Summary The entry of SARS-CoV-2 in permissive cells is mediated by the binding of its spike to angiotensin-converting enzyme 2 (ACE2) on the cell surface. To select ligands able to block this interaction, we screened a library of phages encoding artificial proteins (named αReps) for binding to its receptor binding domain (RBD). Two of them were able to bind the RBD with high affinity and block efficiently the virus entry in cultured cells. Assembled αReps through covalent or non-covalent linkages blocked virus entry at lower concentration than their precursors (with around 20-fold activity increase for a trimeric αRep). These αReps derivates neutralize efficiently SARS-CoV-2 β, γ, δ and Omicron virus variants. Instillation of an αRep dimer in the nasal cavity effectively reduced virus replication in the hamster model of SARS-CoV-2 and pathogenicity. IgG domain able to inhibit SARS-CoV-2 infection have been produced and tested for systemic 66 treatments, but their efficacy by delivery in the nose may not be optimal, due to a poor stability 67 in the nasal cavity environment. Their firmness upon nebulization and aerosolization will be 68 also a main issue for their use as therapeutics. Furthermore, their large-scale production should 69 be economically not affordable in eucaryotic systems and technically difficult to achieve in 70 prokaryotes [4] . 71 As an alternative approach to VHH and antibodies, a family of artificial proteins, named αRep, (Fig. 1) . A large αRep library has been assembled and was demonstrated on a wide 78 range of unrelated protein targets to be a generic source of tight and specific binders. Thus, 79 Reps were previously selected as interactors of HIV-1 nucleocapsid and to negatively interfere 80 with virus maturation [8] . 81 As for all coronaviruses, the SARS-CoV-2 spike (S) protein mediates virus entry to permissive 82 cells. The S protein is a trimeric class 1 fusion protein that binds to its cell receptor, angiotensin 83 converting enzyme 2 (ACE2), before undergoing a dramatic structural rearrangement to fuse 84 the host-cell membrane with the viral membrane [9], [10] . Fusion is triggered when the S1 85 subunit binds to a host-cell receptor via its receptor binding domain (RBD). In order to bind to 86 the receptor, the RBD undergoes articulated movements that transiently expose or hide its 87 surface associated to the binding to ACE2 [11] . The two states are referred to as the "down" 88 and the "up" conformations, in which down corresponds to a state incompetent to receptor 89 binding and up to a state allowing receptor recognition. Due to its key function in the virus 90 cycle, the RBD represents a target to identify binders that block interaction with the host-cell 91 receptor or movements of the RBD between the down to up conformations [12] . 92 Most SARS-CoV-2 infected people presents serum neutralizing antibody activity against the 93 RBD indicating its immunodominance [13] . To reduce antibody binding, the viral evolution 94 has led to the appearance of specific escape mutations in the RBD making current antibody-95 based treatments rapidly less effective [14] . 96 Here, we first obtained a series of Reps specific of the receptor binding domain of the spike 105 An overview of the selection process to generate anti-SARS-CoV-2 Reps specific of the spike 106 is shown in Fig. 1 . In order to select binders blocking SARS-CoV-2 entry into cells, the RBD 107 (amino acids 330 to 550 of the spike S sequence) was used as a bait for screening. The phage 108 display procedure included three rounds of panning followed by a screening step by phage-109 ELISA on individual clones. Nucleotide sequencing allowed the identification of >20 110 independent clones that were retained for further analyses (selected Rep sequences are listed 111 in Fig. S1 ). His-tagged versions of the anti-RBD αRep were expressed in E. coli and purified. 112 We first explored their affinity for the RBD by biolayer interferometry (BLI) at different 113 concentrations to determine their kinetic rate constants. (3 µM) displayed neutralization activity against vesicular stomatitis virus G pseudo-typed 128 MLV, demonstrating their specificity. 129 We confirmed this neutralization activity using SARS-CoV-2 infection of Vero E6 cells (Fig. 130 2D ). C2 showed the highest neutralizing potency with a half-maximal inhibitory concentration 131 (IC50) value of 0.1 µM, while C7 and F9 Reps displayed IC50 values of 4.8 and 11.7 µM, 132 respectively (Fig. 2E) . G1 as well as the anti-influenza H7 Rep did not show neutralization 133 activity. We thus identified three potent neutralizing Reps, with C2 and F9 displaying affinity 134 in the nM range. These two lasts Reps were retained for further analyses. In order to increase avidity and neutralization activity of these RBD binders, we aimed at 138 generating multivalent Reps. We first determined if F9 and C2 recognized non-overlapping 139 binding sites on the RBD to assess their interest to be linked in a fusion protein. Competitive 140 binding assays carried out by BLI showed that C2 and F9 bindings on the RBD did not interfere 141 in a reciprocal manner ( Fig. 3A and 3B) . Competitive binding assays between these two Reps 142 and soluble hACE2 showed that ACE2 binding occurred efficiently after binding of C2 on the 143 RBD. In contrast, binding of F9 on the RBD partially inhibited recognition of hACE2. As a 144 positive control, VHH72 recognizing the receptor binding motif [16] fully blocked hACE2 145 binding on the RBD (Fig. 3C) . These results suggest that the neutralization activity of the C2 146 Rep is not associated to a steric inhibition of the binding of the RBD on ACE2, and that a 147 fusion between C2 and F9 Reps may be synergistic. We thus engineered bivalent Reps 148 constructs using F9 and C2 Reps. We also generated trivalent Reps through the addition of 153 To build the F9-C2 and C2-F9 heterodimers, we inserted a 25 amino acid long flexible linker 154 (GGGGS)5 between these two subunits (Fig. S1 ). This linker length (that can reach 8 nm in 155 length) allows the binding of these heterodimers between adjacent RBDs in the trimer, even in 156 the "up" to "down" spike conformers. To generate the homotrimeric C2-and F9-foldon Reps, 157 the foldon sequence was connected to the C-ter of the αREPs through a 16-amino acid long 158 linker (GSAGSAGGSGGAGGSG) (Fig S1) . These linkers would allow cross-links between 159 spikes at the surface of the virus particle. Unable to express efficiently the C2-F9 construct, 160 only the F9-C2 affinity was characterized by BLI experiments (Fig. 4A) . F9-C2 displayed an 161 equilibrium dissociation constant (KD) of 91 pM, at least three folds better than that of 162 monomers. F9-C2 also showed a substantially slowed dissociation rate constant of 5.86 x 10 -163 5 s -1 owing to enhanced avidity. Circular dichroism revealed melting temperatures of 86.5°, 164 88.3° and 86.0°C for C2, F9 and F9-C2, respectively, confirming the high stability of this class 165 of protein (Fig. S2) . 166 We next investigated the ability of F9-C2 to block RBD-ACE2 interaction by BLI 167 measurements (Fig. 3C) . When F9-C2 was bound to the RBD, addition of ACE2 induced no 168 signal shift demonstrating that F9-C2 dimer is a potent inhibitor of spike binding to ACE2, 169 similarly to the VHH72 [16]. 170 We next explored the neutralization activity of F9-C2 and C2-and F9-foldon for comparison 171 with their parental subunits against SARS-CoV-2 spike pseudo-typed MLV (Fig. 4B) . A 172 synergic effect in neutralisation efficiency was evidenced when the F9 and C2 subunits were 173 covalently linked and when C2 was assembled as a homotrimer. While C2 almost fully blocked 190 In order to evaluate if F9-C2 prophylaxis was effective to limit SARS-CoV-2 infection in vivo, 191 we used Syrian golden hamsters known to reflect the infection in human [18] . We focused on 192 the nasal cavity as we choose to examine how a local treatment could limit the start of the 193 infection in a physiological context. We pre-treated the hamsters with 0.6 mg of F9-C2 194 distributed between the two nostrils 1h prior to infection with 5.10 3 TCID50 of SARS-CoV-2 of 195 the circulating European strain in 2020 (harbouring the D614G mutation in the spike protein) 196 (Fig. 5A) . After such treatment, we observed the presence of infiltrated Reps on the surface 197 of the epithelium layer, indicating an efficient absorption of the molecule (Fig. S3) . The group 198 treated with the non-neutralizing αREP G1 lose weight starting from day 2. Treatment with F9-199 C2 limited weight loss and the difference with G1 treatment almost reach significance at 3 dpi thermostable and can be stored at room temperature, which is a significant advantage for further 294 therapeutic developments. Immunogenicity of the Reps is a potential problem that should be addressed in the future, but To conclude, we selected artificial proteins (Reps) as specific and versatile neutralizing 300 binders targeting the spike of SARS-CoV-2. These biosynthetic proteins provide starting points 301 for SARS-CoV-2 therapeutics able to target emerging variants. With technical optimisation in 302 binder selection and effort to stabilize them in the nasal cavity, we believe that stable 303 proteinaceous inhibitors like Reps and derivates have a real future to threat future pandemics 304 associated to various emerging respiratory viruses. 307 The RBD (223 amino acids starting at position 319 of the spike sequence) coding sequence was 308 cloned in frame behind a sequence encoding a signal peptide and in front of a His-tag coding 309 sequence in the eukaryotic pYD11 expression plasmid. The resulting plasmid was transfected 310 with PEImax (24765-1) (Polysciences, Inc.) in EXPI-293F cells (A14527) (Thermofisher). Transfected cells were then maintained in EXPI expression medium (Gibco, Thermofisher). Selection S1 or RBD Pseudo-types and SARS-CoV-2 neutralization assays In vivo protection assay in hamster Screening phages-aRep aRep linkages aRep production and affinity determination aRep library aRep production and affinity determination * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * * ** ** * Genetics Reveals a Variable Infection Gradient in the Respiratory Tract Massive transient damage of 702 the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in 703 golden Syrian hamsters Antibody therapies could be a bridge to a coronavirus vaccine -but will the world 708 benefit? Nature The αRep 710 artificial repeat protein scaffold: a new tool for crystallization and live cell applications Comparison of ARM and HEAT 713 protein repeats 715 Production and Molecular Structure of a New Family of Artificial Alpha-helicoidal Repeat 716 Proteins (αRep) Based on Thermostable HEAT-like Repeats Alpha-helicoidal 719 HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively 720 Interfere with Viral Genome Packaging and Virus Maturation Distinct conformational states of 723 SARS-CoV-2 spike protein Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease 726 Inhibitor Receptor binding and 728 priming of the spike protein of SARS-CoV-2 for membrane fusion Receptor-binding domain-specific human 731 neutralizing monoclonal antibodies against SARS-CoV and SARS-CoV-2 SARS-CoV-2 734 variants, spike mutations and immune escape Comprehensive 737 mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by 738 polyclonal human plasma antibodies Production of Pseudotyped 741 Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting Structural Basis 744 for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies Structure of bacteriophage T4 fibritin: a 747 segmented coiled coil and the role of the C-terminal domain Pathogenesis and transmission of 750 SARS-CoV-2 in golden Syrian hamsters Mechanisms of SARS-CoV-2 entry into cells Prospects of Neutralizing Nanobodies Against SARS-CoV-2. Front 755 Immunol Integrative overview of antibodies against SARS-CoV-2 and their possible applications 758 in COVID-19 prophylaxis and treatment Antibody therapies could be a bridge to a coronavirus vaccine -but will the world 761 benefit? Nebulised ALX-0171 for respiratory syncytial virus lower respiratory tract infection in 764 hospitalised children: a double-blind, randomised, placebo-controlled, phase 2b trial 767 Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library Processing of gene expression data generated by 771 quantitative real-time RT-PCR Determination of 50% endpoint titer using a simple formula IL-17c is 775 involved in olfactory mucosa responses to Poly(I:C) mimicking virus presence Phylogenetically 778 based establishment of a dengue virus panel, representing all available genotypes, as a tool in 779 dengue drug discovery In vitro screening of a 781 FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication Preclinical evaluation of 784 Imatinib does not support its use as an antiviral drug against SARS-CoV-2 Niclosamide shows strong 787 antiviral activity in a human airway model of SARS-CoV-2 infection and a conserved potency 788 against the Alpha (B.1.1.7), Beta (B.1.351) and Delta variant (B.1.617.2) A pan-serotype 791 dengue virus inhibitor targeting the NS3-NS4B interaction TDPEKVEMYIKNLQDDSMLVRSYAANALGKI GDERAVEPLIKALKDEDLAVRRAAATALGKI GDERAVEPLIKALKDEDSAVRQSAARALGQI GDERAVEPLIKALKDEDPWVRRAAAYALGQI GDERAVEPLIKALKDEDPWVRKTAAEALGKI GDERAVEPLIKALKDEDTNVRYRAAQALGKI GDERAVEPLIKALKDEDAEVRRVAAVALGEI GDERAVEPLIKALKDEDSDVRYGAAVALGQI GGERVRAAMEKLAETGTGFARKVAVNYLETHKSLIS >H12 (n = 5) MRGSHHHHHH TDPEKVEMYIKNLQDDSGHVRVFAAYALGKI GDERAVEPLIKALKDEDSDVRISAANALGKI GDERAVEPLIKALKDEDSAVRQSAAEALGKI GDERAVEPLIKALKDEDSNVRRNAARALGQI GDERAVEPLIKALKDEDAAVRKAAALALGKI GDERAVEPLIKALKDEDSYVRQSAAEALGKI GGERVRAAMEKLAETGTGFARKVAVNYLETHKSLIS >C2F9 MRGSHHHHHHT DPEKVEMYIKNLQDDSVKVRFFAAYALGKI GDERAVEPLIKALKDEDANVRISAAAALGKI GDERAVEPLIKALKDEDAAVRQSAASALGQI GDERAVEPLIKALKDEDENVRREAARALGQI GGERVRAAMEKLAETGTGFARKVAVNYLETHKSLIS GGGGSGGGGSGGGGSGGGGSGGGGS TDPEKVEMYIKNLQDDSVLVRYNAAFALGKI GDERAVEPLIKALKDEDRYVRFSAALALGEI GDERAVEPLIKALKDEDGYVRASAAWALGQI GDERAVEPLIKALKDEDWRVRLSAAKALGKI GDERAVEPLIKALKDEDGEVRVRAANALGKI GDERAVEPLIKALKDEDGYVRRAAAGALGQI GDERAVEPLIKALKDEDWLVRQSAATALGKI GDERAVEPLIKALKDEDPSVRFSAAAALGEI GDERAVEPLIKALKDEDGFVRLSAASALGQI GGERVRAAMEKLAETGTGFARKVAVNYLETHKSLIS >F9C2 MRGSHHHHHHT TDPEKVEMYIKNLQDDSVLVRYNAAFALGKI GDERAVEPLIKALKDEDRYVRFSAALALGEI GDERAVEPLIKALKDEDGYVRASAAWALGQI GDERAVEPLIKALKDEDWRVRLSAAKALGKI GDERAVEPLIKALKDEDGEVRVRAANALGKI GDERAVEPLIKALKDEDGYVRRAAAGALGQI GDERAVEPLIKALKDEDWLVRQSAATALGKI GDERAVEPLIKALKDEDPSVRFSAAAALGEI GDERAVEPLIKALKDEDGFVRLSAASALGQI GGERVRAAMEKLAETGTGFARKVAVNYLETHKSLIS GGGGSGGGGSGGGGSGGGGSGGGGS TDPEKVEMYIKNLQDDSVKVRFFAAYALGKI GDERAVEPLIKALKDEDANVRISAAAALGKI GDERAVEPLIKALKDEDAAVRQSAASALGQI GDERAVEPLIKALKDEDENVRREAARALGQI GGERVRAAMEKLAETGTGFARKVAVNYLETHKSLIS >C2-foldon MRGSHHHHHH T DPEKVEMYI KNLQDDSVKV RFFAAYALGK I GDERAVEPL IKALKDEDAN VRISAAAALG KI GDERAVEP LIKALKDEDA AVRQSAASAL GQI GDERAVE PLIKALKDED ENVRREAARA LGQI GGERVR AAMEKLAETG TGFARKVAVN YLETHKSLIS GSAGSAGGSGGAGGSGYIPEAPRDGQAYVR KDGEWVLLSTFL >F9-foldon MRGSHHHHHH TDPEKVEMYI KNLQDDSVLV RYNAAFALGK I GDERAVEPL IKALKDEDRY VRFSAALALG EI GDERAVEP LIKALKDEDG