key: cord-0886289-erh19k62
authors: Maisonnasse, P; Aldon, Y; Marc, A; Marlin, R; Dereuddre-Bosquet, N; Kuzmina, NA; Freyn, AW; Snitselaar, JL; Gonçalves, A; Caniels, TG; Burger, JA; Poniman, M; Chesnais, V; Diry, S; Iershov, A; Ronk, AJ; Jangra, S; Rathnasinghe, R; Brouwer, PJM; Bijl, TPL; van Schooten, J; Brinkkemper, M; Liu, H; Yuan, M; Mire, CE; van Breemen, MJ; Contreras, V; Naninck, T; Lemaître, J; Kahlaoui, N; Relouzat, F; Chapon, C; Ho Tsong Fang, R; McDanal, C; Osei-Twum, M; St-Amant, N; Gagnon, L; Montefiori, DC; Wilson, IA; Ginoux, E; de Bree, GJ; García-Sastre, A; Schotsaert, M; Coughlan, L; Bukreyev, A; van der Werf, S; Guedj, J; Sanders, RW; van Gils, MJ; Le Grand, R
title: COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models
date: 2021-02-15
journal: Res Sq
DOI: 10.21203/rs.3.rs-235272/v1
sha: 400fab4a0261e7c2b474fa8030df8681e6dd48ba
doc_id: 886289
cord_uid: erh19k62

One year into the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), effective treatments are still needed(1-3). Monoclonal antibodies, given alone or as part of a therapeutic cocktail, have shown promising results in patients, raising the hope that they could play an important role in preventing clinical deterioration in severely ill or in exposed, high risk individuals(4-6). Here, we evaluated the prophylactic and therapeutic effect of COVA1-18 in vivo, a neutralizing antibody isolated from a convalescent patient(7) and highly potent against the B.1.1.7. isolate(8,9). In both prophylactic and therapeutic settings, SARS-CoV-2 remained undetectable in the lungs of COVA1-18 treated hACE2 mice. Therapeutic treatment also caused a dramatic reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg(−1) one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 had a very strong antiviral activity in the upper respiratory compartments with an estimated reduction in viral infectivity of more than 95%, and prevented lymphopenia and extensive lung lesions. Modelling and experimental findings demonstrate that COVA1-18 has a strong antiviral activity in three different preclinical models and could be a valuable candidate for further clinical evaluation.

Across the world, the Coronavirus Disease 19 pandemic caused by severe acute 70 respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to escalate 10 . Despite the 71 progressive rollout of vaccines, there remains an urgent need for both curative and preventive 72 measures, especially in individuals with high risk. Monoclonal neutralizing antibodies (NAbs), 73 isolated from convalescent COVID-19 patients, are one of the most promising approaches and 74 two NAb-based products have already received an emergency use authorization by the FDA. 75 Although their clinical efficacy remains to be fully assessed 4-6 , their capability to reduce viral 76 loads 4,5 shows sufficient promise that such an approach could be effective if the treatment is 77 administered early enough. 78 79 We and others have previously isolated and characterized several highly potent monoclonal 80 NAbs with half-maximum inhibitory concentration (IC50) values in the picomolar range 7,11-14 , 81 with the majority of these targeting the receptor binding domain (RBD) on the S1 subunit of 82 the S protein. We previously identified COVA1-18, an RBD-specific monoclonal Ab, as one 83 of the most potent NAb in vivo 7 . Using three different experimental models as well as 84 mathematical modeling, we demonstrate that its rapid and extensive biodistribution is 85 associated with a very potent antiviral effect, and make it a promising candidate for clinical 86 evaluation, both as a prophylactic or therapeutic treatment of COVID-19. 87 88 COVA1-18 in vitro potency is dependent on avidity 89 To advance our earlier in vitro results 7 on COVA1-18 and allow for better comparability with 90 other studies, we used two new pseudovirus assays, one using lentiviral pseudotypes with an 91 ACE2-expressing 293T cell line 15 , and one using VSV-pseudotypes with Vero E6 cells 16 , to 92 confirm the potency of COVA1-18. With these assays, we confirmed the remarkable potency 93 of COVA1-18 IgG which inhibited lentiviral SARS-CoV-2 pseudovirus with an IC50 of 0.8 ng 94 ml -1 (5.2 pM) and VSV-based pseudovirus with an IC50 of 9 ng ml -1 (60 pM) (Extended Data 95 Fig. 1a , Extended Data Table 1 ). These results were corroborated in multiple independent labs 96 and COVA1-18 was also equipotent against the D614G variant (Extended Data Table 1) Table 1 ). Its Fab displayed a 12-fold weaker binding to RBD 106 compared to IgG (84 nM), with the difference mainly caused by a faster Fab off-rate (Fig. 1a , 107 Extended Data Table 1 ), also observed in a different assay setting (Extended Data Fig. 1d ). 108 With an IC50 of 199 ng ml -1 , the COVA1-18 Fab was 237-fold less potent at neutralizing SARS- 109 CoV-2 pseudovirus, showing that the IgG avidity effect is important for COVA1-18 110 neutralization potency (Extended Data Fig. 1a , Extended Data Table 1 ). 111 112 113 We sought to evaluate whether COVA1-18 could control SARS-CoV-2 viral infection in a 114 previously described Ad5-hACE2 mouse model 22,23 using a 10 mg kg -1 dose. COVA1-18 115 administered intraperitoneally 24 h prior to, or after a SARS-CoV-2 challenge with 10 4 plaque 116 forming units (PFU) was fully protective with no detectable viral replication in the lungs (Fig.   117 1b, c). We then tested the efficacy of COVA1-18 in the golden Syrian hamster model (n = 5 118 per group), which is naturally susceptible to SARS-CoV-2 and develop severe pneumonia upon 119 infection 24 . We evaluated the effect on lung viral loads of 10 mg kg -1 of COVA1-18 given 24 120 h after a 10 5 PFU intranasal challenge (Fig. 1b, d) . At 3 days post-infection (d.p.i.), 3/5 animals 121 had high serum neutralization while 2/5 animals had low neutralization activity (Extended Data 122 Fig. 1e ). On day 3, the COVA1-18 treated group had significantly lower median lung viral 123 titers compared to the control group (3.5 vs 6.7 log10 PFU g -1 , respectively, p<0.01) with lowest 124 viral titers in the higher serum neutralizers (Fig. 1d ). 125 126 127 We evaluated the potential of COVA1-18 to prevent SARS-CoV-2 infection in cynomolgus 128 macaques in a pre-exposure prophylaxis (PrEP) study. The animals were treated intravenously 129 24 h prior to viral challenge with a dose of 10 mg kg -1 of COVA1-18 (Fig. 2a) . Treated and 130 control animals (n = 5 per group) were challenged on day 0 with 10 6 PFU of SARS-CoV-2 via 131 combined intranasal and intratracheal routes using an experimental protocol developed 132 previously 25, 26 . On the day of challenge, the mean COVA1-18 serum concentration was 109 ± 133 2.7 μg ml -1 (Fig. 2b, Extended Data Fig. 2a) , and 4/5 animals had serum neutralization activity 134 while no neutralization activity was observed in the control group (Extended Data Fig. 2b-d) . 135 COVA-18 was also detected in all respiratory tract samples and rectal samples 136 Extended Data Fig. 3a-c) , and represented on average 1.5% and 1.2% of the total IgG in heat-137 inactivated content in the nasopharyngeal and tracheal mucosae, respectively. These levels 138 remained constant throughout the study period and similar levels were detected at 3 d.p.i. in 139 bronchoalveolar lavages (BAL) and saliva ( Fig. 2e-f ). As SARS-CoV-2 can cause damage to 140 non-respiratory organs, we performed a pharmacokinetic study on two additional macaques to 141 characterize the COVA1-18 distribution within the first 24 h (Extended Data Fig. 3d- Following viral challenge, control animals showed similar genomic (g)RNA and subgenomic 149 (sg)RNA levels and kinetics as previously described 25,26 with median peak viral loads (VL) of 150 6.4 and 6.2 log10 copies per ml at 1-2 d.p.i. in the nasopharyngeal and tracheal swabs, were also lower in COVA1-18 recipients compared to controls but the difference did not reach 170 statistical significance (Fig. 3a, b, Extended Fig. 4c ). Overall, these results demonstrate that a 171 10 mg kg -1 dose of COVA1-18 PrEP dramatically reduced the acquisition and/or early spread 172 of SARS-CoV-2 in the different respiratory compartments. Table 2 ). In treated animals, 50 was estimated to 202 2.2 and 0.053 µg ml -1 in the nasopharynx and trachea, respectively, which is roughly 50 and 203 2000 times lower than the plasma drug concentrations of 109.7 µg ml -1 observed on the day of 204 infection (see above). Thus these results confirm that the efficacy of COVA1-18 was very high, 205 with efficacies above 95% and 99.9% in nasopharyngeal and tracheal compartments on the day 206 of infection, respectively (Fig. 4a , Extended Data Fig. 6a ). Given the long half-life of the drug, 207 this efficacy could be maintained over time, and we estimated that the mean individual efficacy 208 of the COVA1-18 in the first 10 days following infection ranged between 96.67% and 97.50% 209 in the nasopharynx and between 99.91% and 99.94% in the trachea (Extended Data Table 3) . In conclusion, our COVA1-18 in vitro data translated into a powerful protective drug in three 267 preclinical models to prevent SARS-CoV-2 replication. Together with our prediction model, 268 these data showed that COVA1-18 could be used in patients at low doses either to prevent 269 infection or to reduce viral loads in a therapeutic setting, with a potential greater impact in 270 high-risk patients. The high in vivo efficacy of COVA1-18 and its demonstrated potency 271 against the B.1.1.7. isolate also suggests it is a great candidate for a NAb cocktail. Ten female cynomolgus macaques were randomly assigned between the control and treated 531 groups to evaluate the efficacy of COVA1-18 prophylaxis. The treated group (n = 5) received 532 one bolus dose of COVA-18 human IgG1 monoclonal antibody (10 mg kg -1 ) by the intravenous 533 route in the saphenous vein one day prior to challenge, while control animals (n = 5) received 534 no treatment. All animals were then exposed to a total dose of 10 6 PFU of SARS-CoV-2 535 (BetaCoV/France/IDF/0372/2020; passaged twice in VeroE6 cells) via the combination of 536 intranasal and intratracheal routes (day 0), using atropine (0.04 mg kg -1 ) for pre-medication 537 and ketamine (5 mg kg -1 ) with medetomidine (0.05 mg kg -1 ) for anesthesia. Animals were 538 observed daily and clinical exams were performed at baseline, daily for one week, and then 539 twice weekly, on anaesthetized animals using ketamine (5 mg kg -1 ) and metedomidine (0.05 540 mg kg -1 ). Body weight and rectal temperature were recorded and blood, as well as 541 nasopharyngeal, tracheal and rectal swabs, were collected. Broncho-alveolar lavages (BAL) 542 were performed using 50 ml sterile saline on 3 d.p.i. Chest CT was performed at 3 d.p.i. in 543 anesthetized animals using tiletamine (4 mg kg -1 ) and zolazepam (4 mg kg -1 ). Blood cell counts, 544 haemoglobin and haematocrit were determined from EDTA blood using a DHX800 analyzer 545 (Beckman Coulter). 

Cross-Neutralization of a SARS-CoV-2 Antibody to a Functionally Conserved 611

Clinical and virological data of the first cases of COVID-19 in Europe: a 613 case series

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Virological assessment of hospitalized patients with COVID-2019

Time course of lung changes at chest CT during recovery from Coronavirus 619 disease 2019 (COVID-19)

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 621 genome sequencing using nanopore

In Vivo Retargeting of Adenovirus Type 5 to αvβ6 Integrin Results in 623

Reduced Hepatotoxicity and Improved Tumor Uptake following Systemic Delivery

Ad5:Ad48 Hexon Hypervariable Region Substitutions Lead to Toxicity and 626

Increased Inflammatory Responses Following Intravenous Delivery

Influenza A Virus Infection in Humans

Zika plasma viral dynamics in nonhuman primates provides insights into early 631 infection and antiviral strategies

Modeling within-host dynamics of influenza virus infection including 633 immune responses

Pharmacodynamic Models implemented in the PFIM software

Parameter estimation in nonlinear mixed effect models 637 using saemix, an R implementation of the SAEM algorithm

Convergence of a stochastic approximation version of 639 the EM algorithm

Representative of 3 independent experiments. (b) Study design with n = 5 per group, 646 except mouse control group (n = 3). Hamsters were infected with 10 5 PFU on day 0 and treated 647 on day 1. Mice received COVA1-18 24 h prior to or after exposure to 10 4 PFU. Lung viral 648 titers at 3 d.p.i. are shown for mice (c) and hamsters (d)

Figure 2. COVA1-18 serum and mucosal pharmacokinetic in infected cynomolgus 653 macaques. (a) Study design. Two groups of n = 5 were exposed to 10 6 PFU of SARS-CoV-2 654 (intranasal and intratracheal routes)

nasopharyngeal fluid, (d) tracheal fluid (means with range), (e) bronchoalveolar lavage (BAL) 658 and (f) saliva (means ± SEMs). The red dashed line indicates challenge day

Figure 3. COVA1-18 pre-exposure prophylaxis protects cynomolgus monkeys against 662

Medians with range are indicated for fluids and bars 665 represent medians for BAL. (c) Chest CT scores were determined at 3 d.p.i. and at 2 or 5 d.p.i 666 for historical controls

Individual prediction of 671 the nasopharyngeal gRNA and sgRNA in control (top) and treated animals (bottom) with 672 individual efficacy prediction indicated (green line). The dashed red line indicates the time of 673 infection. gRNA (squares) and sgRNA (circles) data are indicated as plain (above LoQ) or open 674 (below LoQ). (b) Model predictions of gRNA and sgRNA dynamics with 4 doses of COVA1-675 18 given 24 h prior to challenge (arrow). (c) Simulation as in (b) with COVA1-18 given 24 h 676 post-infection. Black dotted lines indicate LoQ (limit of quantification)

Extended Data Figure 1. COVA1-18 IgG and Fab neutralization, cross-reactivity, 680 binding kinetic and Syrian hamster serum neutralization. (a) IgG (grey) and Fab (black

Representative of n ≥ 4 independent 682 experiments. (b) Antigen specificity of COVA1-18 was assessed by ELISA against the soluble 683 S protein derived from different human coronaviruses. (c) BLI sensorgrams of COVA1-18 684 binding to immobilized soluble SARS-CoV-2 S protein. Representative of n ≥ 2 independent 685 experiments. (d) BLI sensorgrams of COVA1-18 binding to SARS-CoV-2 RBD loaded onto 686 the sensor chip at various concentrations

Syrian hamsters for the control group (left) and COVA1-18 treated group

Extended Data Figure 2. Serum and mucosal pharmacokinetics of COVA1-18 in treated 691 macaques (1/2). (a) Serum COVA1-18 concentration for each animal. The mean COVA-18 692 concentration for each group is indicated by a thick blue line (treated animals) and a thick black 693 line (control). (b) Individual serum neutralization ID50. (c) Serum neutralization curve for each 694 animal at the indicated day post-treatment. (d) Individual serum neutralization ID50 with titer 695 range indicated as ID50 21-49 in green

Extended Data Figure 3. Serum and mucosal pharmacokinetics of COVA1-18 in treated 698 macaques (2/2). The COVA1-18 concentrations measured in nasopharyngeal (a), tracheal (b) 699 and rectal (c) fluids by ELISA are reported for each animal in both groups. (d) Serum COVA1-700 18 concentration from two animals injected with 10 mg kg -1 of COVA1-18 and

The two macaques were euthanized at 702 24 h post-treatment and organs analyzed to assess the biodistribution of COVA1-18. The 703 concentration of COVA1-18 was normalized to the weight of each sample for every organ

COVA1-18 was measured in fluid samples of the PK study animals and normalized to the total 705 cynomolgus IgG content for each sample. LoQ, limit of quantification

cynomolgus 708 monkeys against SARS-CoV-2 challenge and clinical symptoms. (a) Genomic (g)RNA and 709 (b) subgenomic (sg)RNA loads determined by PCR in nasopharyngeal fluids (left) and tracheal 710 fluids (right) of control (top) and treated (bottom) animals. (c) gRNA (top) and sgRNA 711 (bottom) in the bronchoalveolar lavages (BAL) at day 3 post-infection

lymphocyte count in the blood of control (top) and treated (bottom) animals. LoD, limit of 713 detection

Extended Data Figure 5. Sequences in treated and exposed NHP. Viral population 716 sequences in the nasopharyngeal swabs at day 3 were analyzed by Next Generation 717

Variants count detected in the N and ORF1ab genes for each individual (left) 718 and cumulative variants count for each gene in the control and COVA1-18 treated groups 719 (right). (b) Individual (left) and cumulative (right) synonymous and missense variants count 720 for the control and treated groups. (c) Nucleotide substitution observed by type for both groups

Extended Data Figure 6. Modeling of viral dynamics and treatment efficacy (1/2). (a)

The dashed red line 725 indicates the time of viral infection. gRNA (squares) and sgRNA (circles) data are indicated as 726 plain (above LoQ) or open (below LoQ). (b) Individual prediction of the COVA1-18 plasma 727 concentration. (c-d) Simulation of the predicted gRNA (top) and sgRNA (bottom) viral loads 728 in the nasopharynx

COVA1-18 given 24 h prior challenge (arrow). (e-f) Simulation as in (c) with COVA1-18 given 730 24 h post-infection. Black dotted lines indicate the limit of quantification (LoQ). i.v., 731 intravenous

Extended Data Figure 7. Modeling of viral dynamics and treatment efficacy (2/2)

Simulation of the predicted gRNA (top) and sgRNA (bottom) viral loads in the nasopharynx, 735 according to the dose of COVA1-18 received and the dose of virus received

Exposure Prophylaxis (PrEP) treatment at -1 d.p.i., viral load measured at 2 d.p.i.; Right: 737 Therapeutic treatment at 1 d.p.i., viral load measured at 3 d.p.i. Black: control; yellow: 0.1 mg 738 kg -1 ; green: 1 mg kg -1 : orange: 5 mg kg -1

Extended Data Table 1. BLI and neutralization potency of IgG vs Fab in HEK293T 740 hACE2 cells. AMC and Duke neutralization assays use lentiviral pseudotyped particles and 741 HEK293T hACE2 cells. Nexelis neutralization assay uses VSVΔG

Vero E6 cells. BLI, biolayer interferometry; RBD, receptor binding domain

Extended Data Table 2. Parameter estimates of the viral dynamic model

Extended Data Table 3. Mean individual efficacy of the COVA1-18 for each individual in 748 both compartments (calculated over the first 10 days of administration)

Acknowledgments: We thank Benoit Delache

Tierry Prot 753 and Christina Dodan for their help with the macaque experiments

Laurine Moenne-Loccoz and Julie Morin for the RT-qPCR, and for the preparation of 755 reagents

Blanche Fert for her help with the CT scans

Elodie Guyon for the macaque sample processing

Sylvie Keyser for the transports 757 organization; Nastasia Dimant and Brice Targat for their help with the experimental studies in 758 the context of COVID-19-induced constraints; Frédéric Ducancel and Yann Gorin for their 759 help with the logistics and safety management

We thank Sylvie Behillil and Vincent Enouf for contribution to viral stock 761 challenge production, Antoine Nougairede for sharing the plasmid used for the sgRNA assays 762 standardization and Paul Bieniasz for donating cells and reagents for pseudovirus neutralization 763 assays. We thank Matt Hyde and Julie Williams

We 765 acknowledge support from CoVIC supported by the Bill and Melinda Gates Foundation. We 766 thank staff at the ISMMS CCMS vivarium for their assistance. We also thank Randy Albrecht 767 for support with the BSL-3 facility and procedures at the ISMMS and Richard Cadagan for

Funding: This study was supported by the Netherlands Organization for Scientific Research 771 (NWO) Vici grant (to R.W.S.), the Bill & Melinda Gates Foundation through the Collaboration 772 for AIDS Vaccine Discovery (CAVD) grant INV-002022

is a recipient of an AMC Fellowship, Amsterdam UMC and a COVID-19 775 grant of the Amsterdam Institute of Infection and Immunity, the

are recipients of support from the University of Amsterdam Proof of Concept fund 777 (contract no 200421) as managed by Innovation Exchange Amsterdam (IXA)

Disease Models and Innovative Therapies (IDMIT) research infrastructure is supported by the 779 'Programme Investissements d'Avenir, managed by the ANR under reference

The Fondation Bettencourt Schueller and the Region Ile-de-France contributed to the 781 implementation of IDMIT's facilities and imaging technologies. The NHP study received 782 financial support from REACTing, the Fondation pour la Recherche Médicale

AM-CoV-Path) and the European Infrastructure TRANSVAC2 (730964)

), by supplements to NIAID grant U19AI135972 and DoD 789 grant W81XWH-20-1-0270, by the Defense Advanced Research Projects Agency (HR0011-790 19-2-0020), and by the generous support of the JPB Foundation, the Open Philanthropy Project 791 (research grant 2020-215611 (5384) and anonymous donors to A.G.S. Part of this study was 792 supported by the Bill and Melinda Gates Foundation through grants OPP1170236 and INV-793 004923 (I.A.W.) and through the Global Health Vaccine Accelerator Platforms (GH-VAP) and 794 the Coronavirus Immunotherapy Consortium (CoVIC) (Nexelis)

A.K. designed, performed and analysed the hamster experiment. M.S. 802 designed, performed and analysed the mouse experiment. A.W.F. performed the mouse 803 experiment. J.L.S. produced antibodies and performed ELISAs. A.G. contributed to the 804 predictive model development

conceived, designed, supervised the project, acquired funding, provided resources 818 and wrote the manuscript

The data that support the findings of this study are available from the corresponding author 832 upon reasonable request. 833 Correspondence and requests for materials should be addressed to roger.le-grand@cea.fr