Antonio Gonçalves

Julie Bertrand

Ruian Ke

Emmanuelle Comets

Xavier De Lamballerie

Denis Malvy

Andrés Pizzorno

Olivier Terrier

Manuel Rosa

Timing of Antiviral Treatment Initiation is Critical to Reduce SARS-CoV-2 Viral Load

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ARTICLE Timing of Antiviral Treatment Initiation is Critical to Reduce SARS-CoV-2 Viral Load

Antonio Gonçalves 1* , Julie Bertrand 1 , Ruian Ke 2 , Emmanuelle Comets 1 , Xavier de Lamballerie 3 , Denis Malvy [START_REF] Zou | SARS-CoV-2 viral load in upper respiratory specimens of infected patients[END_REF][START_REF] Lauer | The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application[END_REF] , Andrés Pizzorno [START_REF] Best | Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies[END_REF] , Olivier Terrier [START_REF] Best | Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies[END_REF] , Manuel Rosa Calatrava [START_REF] Best | Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies[END_REF] , France Mentré 1 , Patrick Smith [START_REF] Banerjee | Estimating biologically relevant parameters under uncertainty for experimental within-host murine West Nile virus infection[END_REF] , Alan S. Perelson [START_REF] Friberg | Acute bacterial or viral infection-What's the difference? A perspective from PKPD modellers[END_REF] and Jérémie Guedj 1, * We modeled the viral dynamics of 13 untreated patients infected with severe acute respiratory syndrome-coronavirus 2 to infer viral growth parameters and predict the effects of antiviral treatments. In order to reduce peak viral load by more than two logs, drug efficacy needs to be > 90% if treatment is administered after symptom onset; an efficacy of 60% could be sufficient if treatment is initiated before symptom onset. Given their pharmacokinetic/pharmacodynamic properties, current investigated drugs may be in a range of 6-87% efficacy. They may help control virus if administered very early, but may not have a major effect in severely ill patients.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, China, has become a global pandemic. By May 7, 2020, this virus had infected more than 3,700,000 people worldwide and caused more than 260,000 deaths. To readily propose a first line of defense and combat the virus in hospitalized patients, the World Health Organization relies on already existing drugs ("repurposed") that are immediately available in large quantities and have a good safety profile. In coordination with other European institutions, France is implementing a randomized clinical trial in hospitalized patients ("DisCoVery," NCT04315948) comparing the efficacy of lopinavir/ritonavir ± IFN-β-1a, remdesivir, and hydroxychloroquine (HCQ) in hospitalized patients. However, the clinical efficacy of currently available therapies is unknown and could be limited. [START_REF] Cao | A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19[END_REF] Here, we fit mathematical models of viral dynamics to in vivo data to estimate parameters driving viral replication. We then use these models to predict the needed efficacy of treatments. [START_REF] Friberg | Acute bacterial or viral infection-What's the difference? A perspective from PKPD modellers[END_REF] By combining the expected drug concentrations and half-maximal effective concentration (EC 50 ) of drug candidates, we also use the model to predict the effects of various dosing regimens (doses and timing of treatment initiation) on viral load dynamics.

METHODS

Data used for fitting

We used published data from 13 untreated patients infected with SARS-CoV-2 that were followed in 4 Singapore hospitals. [START_REF] Young | Epidemiologic features and clinical course of patients infected with SARS-CoV-2 in Singapore[END_REF] Patients were hospitalized in median at day 3 after onset of symptoms (range 1-10 days) and had a median symptomatic period of 12 days (range 5-24 days). Viral loads in nasopharyngeal swabs were measured by real-time reverse transcriptase polymerase chain reaction (lower limit of quantification: 38 cycles-threshold, CT) at multiple time points with an observed peak of viral load at day 5 post onset of symptoms (range 2-27 days).

1 Université de Paris, IAME, INSERM, Paris, France; 2 Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, USA; 3 Institut Hospitalo-Universitaire Méditerranée Infection, UMR "Emergence des Pathologies Virales" (EPV: Aix-Marseille University -IRD 190 -Inserm 1207 -EHESP), Marseille, France; [START_REF] Zou | SARS-CoV-2 viral load in upper respiratory specimens of infected patients[END_REF] Inserm, UMR 1219, Université de Bordeaux, Bordeaux, France; [START_REF] Lauer | The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application[END_REF] Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France; 6 CIRI, Centre International de Recherche en Infectiologie, (Team VirPath), Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Lyon, France; [START_REF] Banerjee | Estimating biologically relevant parameters under uncertainty for experimental within-host murine West Nile virus infection[END_REF] Certara, Integrated Drug Development, Princeton, New Jersey, USA. *Correspondence: Antonio Gonçalves (antonio.goncalves@inserm.fr) and Jérémie Guedj (jeremie. guedj@inserm.fr) Received: May 15, 2020; accepted: June 12, 2020. doi:10.1002/psp4.12543

Study Highlights

WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? ✔ Repurposed drugs are being evaluated in clinical trials but little is known about their efficacy on severe acute respiratory syndrome-coronavirus 2 viral kinetics. WHAT QUESTION DID THIS STUDY ADDRESS? ✔ Our study aims to combine pharmacokinetic/pharmacodynamic and viral kinetics modeling to anticipate the effects of lopinavir/ritonavir, hydroxychloroquine (HCQ), IFN-β -1a, and remdesivir.

WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

✔ Given the predicted efficacy of lopinavir/ritonavir, HCQ, IFN-β -1a, and remdesivir, it is unlikely that these drugs will have a major effect on viral kinetics if they are administered as monotherapy after symptom onset. HOW MIGHT THIS CHANGE DRUG DISCOVERY, DE-VELOPMENT, AND/OR THERAPEUTICS? ✔ Our results suggest that these drugs should be evaluated in persons exposed to the virus but prior to appearance of the first symptoms.

Data presented in CT were transformed to log 10 copies/ mL using a published relationship in Zou et al. [START_REF] Zou | SARS-CoV-2 viral load in upper respiratory specimens of infected patients[END_REF] and the model was fit to the log 10 viral load. Of note, the transformation from CT to log 10 copies/mL does not affect the estimates of parameters of interest, in particular R 0 and the death rate of productively infected cells. Time since infection was assumed to be 5 days before the onset of symptoms. [START_REF] Lauer | The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application[END_REF] In a sensitivity analysis, we also examined values of 2 and 10 days.

Model

Viral dynamics was fitted using a target cell limited model with an eclipse phase.

The model considers three populations of cells: target cells, T, infected cells in the eclipse phase, I 1 , and productively infected cells, I 2 . Given the timescale of the infection, we neglect target cell proliferation and natural death, and we focused on the process of cell depletion by virus infection. We assumed target cells become infected with rate constant β. After an average time of 1/k, these cells start producing virus and are cleared with per capita rate δ. Virions are released from productively infected cells I 2 at rate p per cell and are cleared from the circulation at per capita rate c or lost by infecting a target cell. Based on this model, the basic reproduction number, R 0 , the average number of cells infected by a single infected cell at the beginning of the infection, is [START_REF] Best | Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies[END_REF][START_REF] Banerjee | Estimating biologically relevant parameters under uncertainty for experimental within-host murine West Nile virus infection[END_REF] To determine the target cell concentration, the following calculation was done. We assumed that the total number of epithelial cells in the upper respiratory tract was 4 × 10 8 cells, distributed in a volume of 30 mL. [START_REF] Baccam | Kinetics of influenza A virus infection in humans[END_REF] Assuming that 1% of these cells express the ACE2 receptor and associated proteases needed for viral entry, [START_REF] Muus | Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells[END_REF] the target cell concentration, T 0 , was fixed to 1.33 × 10 5 cells/mL. Following what was found in other viral infections, including acute infection, [START_REF] Best | Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies[END_REF] the clearance rate of virus, c, was assumed to be fast and equal to 10 day -1 but values of 5 and 20 day -1 were also evaluated.

Model building strategy

Because not all parameters can be identified when only viral load data are available, parameters V 0 and k were fixed at 10 -1 copies/mL and 3 day -1 , respectively, which corresponds to an initiation of the viral production 8 hours after cell infection on average. [START_REF] Agostini | Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease[END_REF] A sensitivity analysis was also performed with different values of k = {1, 3, 5} day -1 and V 0 = {10 -3 , 10 -2 , 10 -1 } copies/mL to assess the robustness of the parameter estimates. The parameter R 0 was estimated instead of the infection rate β by a change of variables in Eq. 1.

Parameters were estimated in a nonlinear mixed-effect modeling framework using the SAEM algorithm implemented in Monolix (www.lixoft.com). The model providing the best description of the data was used for the predictions and the individual data fitting, and model averaging was used to correct for the model uncertainty when calculating confidence intervals of estimated parameters. [START_REF] Gonçalves | Model averaging in viral dynamic models[END_REF] Predicting the effects of treatment according to the antiviral efficacy and the time of treatment initiation We assumed that antivirals with a constant effectiveness ε could reduce R 0 by a factor (1-ε), with ε taking values from 50-99% in Eq. 2. We considered different times of treatment initiation, from the time of infection to 3 days after the symptom onset. For each treatment strategy, we calculated the reduction in viral load at the peak of infection in the absence of treatment (i.e., 5 days after symptom onset).

Model including an innate immune response

We also examined the possibility that cell infection is limited by an innate immune response that renders cells refractory to infection, as was proposed for other acute viral infections. [START_REF] Baccam | Kinetics of influenza A virus infection in humans[END_REF][START_REF] Pawelek | Modeling within-host dynamics of influenza virus infection including immune responses[END_REF] In this model, two additional compartments are added, one for a cytokine (e.g., IFN) released in response to antigen, and one representing cells in an antiviral state that cannot be infected (Supplementary Information). Pharmacokinetic/pharmacodynamic drug properties of lopinavir/ritonavir, HCQ, IFN-β-1a, and remdesivir We relied on the literature to find pharmacokinetic (PK) population models and parameter values of lopinavir/ritonavir, [START_REF] Wang | Integrated population pharmacokinetic/viral dynamic modelling of lopinavir/ritonavir in HIV-1 treatment-naïve patients[END_REF] plasma HCQ, [START_REF] Morita | Population pharmacokinetics of hydroxychloroquine in Japanese patients with cutaneous or systemic lupus erythematosus[END_REF] IFN-β-1a, [START_REF] Hu | COMPARE: Pharmacokinetic profiles of subcutaneous peginterferon beta-1a and subcutaneous interferon beta-1a over 2 weeks in healthy subjects: pharmacokinetics of peginterferon beta-1a and s.c. interferon beta-1a[END_REF] and remdesivir, as well as reported EC 50 values in vitro (see Table 1). For lopinavir EC 50 , specific results were obtained as follows. Vero E6 cells were infected by SARS-CoV-2 (strain BetaCoV/ France/IDF0571/2020) at a multiplicity of infection of 0.01 and treated with several concentrations of lopinavir 1 hour after infection. Supernatant samples were collected at 48 and 72 hours postinfection. Relative quantification of viral genome was performed by real-time quantitative polymerase chain reaction RT-qPCR from RNA extracted using QIAamp viral RNA Mini Kit (Qiagen). IC 50 values of lopinavir (5.246 μM and 4.941 μM at 48 and 72 hours postinfection, respectively) were calculated from dose-response curve using a four-parameter logistic regression model.

To determine the mean antiviral efficacy of these drugs, we simulated their plasma PK profiles considering clinical regimens used in the Discovery trial, namely 400-100 mg twice daily (b.i.d.) for lopinavir/ritonavir, 400 mg b.i.d. the first day (loading dose) followed by 400 mg once daily (q.d.) for HCQ and 12 MIU for IFN-β-1a. For each regimen, we simulated 100 PK profiles according to the reported parameter distributions. Then, we calculated for each simulated individual the mean inhibitory coefficient, sometimes called the mean antiviral effectiveness,

= 1 7 × ∫ 7 0 C(u) C(u)+EC 50
du during the first week of treatment, and the mean value over the N profiles are given in Table 1. For comparison purposes, we based the analysis ( 1)

dT dt = -VT dI 1 dt = VT -kI 1 dI 2 dt = kI 1 -I 2 dV dt = pI -cV -VT (2) R 0 = p T 0 (c + T 0 )
on total plasma concentrations and did not adjust for plasma protein binding when computing efficacy.

Because the PK parameters of remdesivir have not been reported yet in humans, the same method could not be applied. We used the summary statistics reported in the summary for compassionate use of remdesivir filed by Gilead to the European Medicines Evaluation Agency (EMEA) [START_REF] Francisco | EMA provides recommendations on compassionate use of remdesivir for COVID-19[END_REF] to derive the mean concentration of the active metabolite in peripheral blood mononuclear cell, mean serum concentration (C mean ), using the area under the curve (AUC) after a loading dose of 200 mg and after repeated doses of 100 mg q.d., and we assumed that the EC 50 for the metabolite was equal to the EC 50 of the parent. [START_REF] Pizzorno | Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia[END_REF]

RESULTS

We used a "target-cell limited" model with an eclipse phase. [START_REF] Baccam | Kinetics of influenza A virus infection in humans[END_REF] given by Eq. 1 to characterize the viral load dynamics of 13 hospitalized patients in Singapore for which data obtained from frequent nasopharyngeal swabs were available. [START_REF] Young | Epidemiologic features and clinical course of patients infected with SARS-CoV-2 in Singapore[END_REF] Because this model needs to incorporate a date of infection, an incubation period of 5 days was used as the most plausible date of infection in each patient. [START_REF] Lauer | The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application[END_REF] The model fitted the data well (Figure 1) and using a model-averaging approach to take into account model uncertainty, [START_REF] Gonçalves | Model averaging in viral dynamic models[END_REF] the within-host basic reproductive number, R 0 , was found equal to 8.6 (95% confidence interval (CI) = 1.9-17.6), and the death rate of productively infected cells was estimated as 0.60 day -1 (95% CI = 0.22-0.97), corresponding to a median half-life of 1.2 days (Table 2 and Figure S1). In influenza A, another respiratory infectious disease, estimates of the within host R 0 varied greatly, but the half-life of infected cells was shorter than 10 hours (see more details in ref. 18), suggesting a faster clearance of influenza-infected cells than SARS-CoV-2. The viral production rate p was also [START_REF] Hu | COMPARE: Pharmacokinetic profiles of subcutaneous peginterferon beta-1a and subcutaneous interferon beta-1a over 2 weeks in healthy subjects: pharmacokinetics of peginterferon beta-1a and s.c. interferon beta-1a[END_REF] 175 IU/mL [START_REF] Woelfel | Clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster[END_REF] 12 MIU at D0, D2, D5 18%

Remdesivir EMEA guidelines [START_REF] Francisco | EMA provides recommendations on compassionate use of remdesivir for COVID-19[END_REF] 1 µM 17 200 mg q.d. at D0, followed by 100 mg q.d.

87%

We assume that the total concentrations were the driver of efficacy, and we did not consider intracellular metabolites or free drug concentrations. D, day; EC 50 , half-maximal effective concentration; EMEA, European Medicines Evaluation Agency; PK/PD, pharmacokinetic/pharmacodynamic. 2). However, as shown previously, p cannot be uniquely identified unless the initial target cell density T 0 is known. [START_REF] Miao | On identifiability of nonlinear ode models and applications in viral dynamics[END_REF][START_REF] Stafford | Modeling plasma virus concentration during primary HIV infection[END_REF] Therefore, the only quantity that can be reliably estimated is the product p × T 0 , equal to 3.0 × 10 6 copies/day (95% CI = 0-7.9 × 10 6 ]). Parameter estimates and CIs were also consistent across models assuming a viral clearance c of 5 or 20 day -1 (see Table S1).

Our model, along with the parameter estimates given above, also inform us both on the time to initiate antiviral treatment, and the level of efficacy that needs to be achieved to reduce viral load. [START_REF] Best | Zika plasma viral dynamics in nonhuman primates provides insights into early infection and antiviral strategies[END_REF] As limited information is available on the mechanisms leading to viral clearance, and how they may be modulated by treatment, we used our model to predict the effects of treatment at day 5 post-symptoms, which corresponds to the time the viral load tends to peak in the absence of treatment in these data. [START_REF] Young | Epidemiologic features and clinical course of patients infected with SARS-CoV-2 in Singapore[END_REF] We considered a simple case where the drug effectiveness is assumed to be constant after therapy initiation (see Methods) and we calculated the minimal efficacy that would be needed to generate more than 2 logs of viral decline at peak viral load in the 13 studied patients (Figure 2). As predicted by viral kinetic modeling theory, 2 we found that the impact of treatment on peak viral load is inversely correlated with the time of treatment initiation. For a putative treatment blocking the viral production p and initiated at the time of infection, symptom onset, or 3 days post-symptom onset, a median efficacy of at least 60%, 90%, and 99% in reducing viral replication would be needed, respectively, to generate more than 2 logs of decline in the peak viral load (Figure 2). The results obtained assuming 2 or 10 days of incubation are presented in Figures S2 andS3. We also considered the case of a drug like hydroxychloroquine blocking viral infection (parameter β in Eq. 1). Results were similar to those obtained before, as long as the treatment was initiated before or at symptom onset. However, initiating a treatment 3 days post-symptom onset could not reduce peak viral load, regardless of the drug effectiveness (see Figure S4). The model, including an innate immune response, did not improve the data fitting and, therefore, was not selected for inclusion in the main text. However, the same analysis on viral dynamics and treatment was conducted and the conclusions remained unchanged (see Figures S5, S6 and Table S2).

How do these levels of effectiveness compare with the antiviral drugs that are currently being investigated? To study this question, we assumed that the treatment antiviral effectiveness at time t after treatment initiation, (t), was re- lated to the plasma total drug concentration, C(t): (t) = C(t) C(t)+EC 50 (except for remdesivir, see Methods) and the mean antiviral effectiveness during the first 7 days of treatment is given by =

1 N × 1 7 × ∫ 7 0 C(u) C(u)+EC 50
du . Given their PK and pharmacodynamic properties (Table 1), we calculated a mean antiviral effectiveness of up to 66% for lopinavir/ritonavir, 6% for hydroxychloroquine, 18% for IFN-β-1a, and 87% for remdesivir. Given these estimates, these compounds are unlikely to have a dramatic effect on peak viral load if administered after the onset of symptoms. In fact, the effective concentrations will presumably be lower in patients, as drug availability may be further limited by protein binding (in particular for lopinavir, which has a protein binding rate > 98%) or capability to penetrate respiratory compartments. Importantly, levels of antiviral efficacy of ~ 50% could nonetheless be relevant in a prophylactic setting, before symptom onset, to reduce viral replication in the upper respiratory tract and reduce the risk of large infiltration to the lungs before an effective immune response is mounted to clear the virus. [START_REF] Friberg | Acute bacterial or viral infection-What's the difference? A perspective from PKPD modellers[END_REF] Note, above, we calculated the effectiveness of drugs administered in monotherapy for their usual dosing regimen. We did not consider drugs that could directly target infected cells and lead to their elimination, such as some monoclonal antibodies.

DISCUSSION

Overall, our results emphasize that the PK/pharmacodynamic properties of lopinavir/ritonavir, HCQ, IFN-β-1a, and remdesivir make them unlikely to have a dramatic impact on nasopharyngeal viral load kinetics if they are administered after symptom onset. However, these drugs may be relevant to reduce viral replication if administered early (i.e., as a pre-exposure or post-exposure prophylaxis). This conclusion, however, depends on a number of hypotheses that we discuss below.

First, we focused on the capability of drugs to reduce the peak viral load making the implicit hypothesis that reducing peak viral load would likely reduce symptoms and disease severity. However, the relationship between viral kinetics and disease severity is still debated, with several studies suggesting that viral load did not differ between patients with severe and nonsevere disease. [START_REF] Zheng | Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China[END_REF][START_REF] Liu | Serum hepatitis B virus RNA: a new potential biomarker for chronic hepatitis B virus infection[END_REF] Related to this question, our study had some limitations. Our calculations relied on blood or plasma drug concentrations. Except for HCQ, for which the ratio of lung-to-plasma concentration is known to be high, [START_REF] Yao | In vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)[END_REF][START_REF] Maisonnasse | Hydroxychloroquine in the treatment and prophylaxis of SARS-CoV-2 infection in non-human primates[END_REF] the lung exposure of the other drugs that we considered is unknown, and their effect on viral load in the lower respiratory tract, as measured from broncho-alveolar aspirates, for instance, may differ. In addition, the drug EC 50 that we used were determined on Vero E6 cells, an in vitro system that may not reflect the in vivo EC 50 . For instance, we found in another study that HCQ had no antiviral activity in a more physiological model of reconstituted human airway epithelium, and this may explain the absence of antiviral of activity of HCQ in vivo against SARS-CoV-2. [START_REF] Maisonnasse | Hydroxychloroquine in the treatment and prophylaxis of SARS-CoV-2 infection in non-human primates[END_REF][START_REF] Boulware | A randomized trial of hydroxychloroquine as postexposure prophylaxis for Covid-19[END_REF] Finally, we focused solely on the antiviral effects of these drugs, and did not consider other potential effects of these drugs, such as their immunomodulatory effects. Such effects have been suggested for drugs that are not purely antivirals, such as HCQ and IFN-β-1a. [START_REF] Liu | Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro[END_REF][START_REF] Ingraham | Immunomodulation in COVID-19[END_REF] Another implicit implication of our work is the benefit of drugs used for prophylaxis (i.e., before exposure to the virus). In that case, the objective of the treatment may be to "flatten the peak viral load" (by analogy with the now popular terminology of epidemiological models) but also to prevent infection. Our deterministic, ordinary differential equation-based model, cannot reproduce virus extinction, but this can be captured using a stochastic version of the same model. [START_REF] Czuppon | Predicted success of prophylactic antiviral therapy to block or delay SARS-CoV-2 infection depends on the targeted mechanism medRxiv[END_REF] Our modeling provides estimates of viral kinetic parameters, in particular R 0 and the loss rate of infected cells, δ, and the limit of extrapolation of these parameters also need to be well understood. The advantage of studying this series of patients was the fact that viral load was sampled extremely frequently and early in the infection. However, the number of patients we studied was quite small (only 13), and the patients were rather young (37 years, range 31-56 years) and may not be representative of patients who typically evolve to have severe forms of the disease. [START_REF] Zheng | Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China[END_REF] Therefore, more data will be needed from various populations to estimate precisely the parameter distribution in the population of patients that are most in need of antiviral therapy. Finally, our estimate of the loss rate of free virus and infected cells are constant over time, which neglects the effects of the adaptive immune response. For instance, antibodies that emerge in the second week after symptom onset, [START_REF] Woelfel | Clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster[END_REF][START_REF] Wu | Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications[END_REF] may contribute to accelerate viral clearance or reduce viral infectivity. However, to include these effects would require more complex models and quantitative data on these antibodies and their in vivo effects, which are currently lacking. Further, our analysis of the antiviral effects of repurposed drugs focused on their ability to reduce the peak viral load, which typically occurs well before the antibody response emerges and, hence, the analysis we presented here should not be affected in any major way by our neglect of antibody responses.

Future models of drug efficacy may need to account for viral resistance, as it is possible that continued viral replication in the presence of drug will select for drug-resistant mutations, [START_REF] Perelson | Combination antiviral therapy for influenza: predictions from modeling of human infections[END_REF] although coronaviruses are unusual in that they appear to have low mutation rates due to RNA proofreading capability. 32 Drug combination therapy and more aggressive dosing, including consideration of loading doses to rapidly achieve therapeutic exposures, may be beneficial to maximize efficacy of these repurposed antiviral agents. [START_REF] Smith | Dosing will be a key success factor in repurposing antivirals for COVID-19[END_REF] For all these reasons, the outcome of randomized clinical trials remains urgently needed, and the analysis of their impacts not only on viral clearance but also on disease severity will be critical to design more potent drugs. Supporting Information. Supplementary information accompanies this paper on the CPT: Pharmacometrics & Systems Pharmacology website (www.psp-journal.com).

Figure 1

 1 Figure1Individual predictions of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) of 13 untreated patients from Young et al.[START_REF] Young | Epidemiologic features and clinical course of patients infected with SARS-CoV-2 in Singapore[END_REF]

Figure 2

 2 Figure 2 Reduction in viral load at day 5 post-symptom onset according to the level of antiviral effectiveness of a treatment blocking the viral production and the timing of treatment initiation (a) at time of infection; (b) at time of symptom onset; (c) 3days after symptom onset). We assumed an incubation period of 5 days.

Funding.

  Antonio Gonçalves is funded by a grant from Roche Pharmaceutical Research and Early Development. The study has received financial support of the national Research Agency (ANR) through the ANR-Flash calls for COVID-19 (TheraCoV ANR-20-COVI-0018). Portions of this work were done under the auspices of the US Department of Energy under contract 89233218CNA000001. We also gratefully acknowledge the support of the US Department of Energy through the LANL/LDRD Program for this work (A.S.P. and R.K.).

Table 1

 1 PK/PD properties of candidate antiviral drugs

	Drug	PK parameter	EC 50	Dosing regimen D0-D7	= 1 N × 1 7 × ∫	7 0	C(u) C(u)+EC 50	du
	Lopinavir/ritonavir	Wang et al. 13	5.2 μM (unpublished)	400/100 b.i.d.	66%
	Hydroxychloroquine	Morita et al. 14	4.2 μM 27	400 mg b.i.d. at D0, followed by 400 mg	6%
				q.d.
	IFN-β-1a	Hu et al.

Table 2

 2 Median and confidence intervals of R 0 , δ, and p across models and following model averaging procedure

	k (day -1 )	V 0 (log 10 cp/mL )	R 0 [95% CI]	δ [95% CI] (day -1 )	p [95% CI] (day -1 )
	1	10 -1	13.1 [5.1-21.8]	0.68 [0.44-0.9]	26.55 [0-57.32]
		10 -2	15.4 [7.9-23.2]	0.71 [0.46-0.94]	32 [8.38-56.26]
		10 -3	19.1 [10.3-28.8]	0.71 [0.46-0.94]	35.18 [12.46-60.06]
	3	10 -1	8.2 [3.5-13.1]	0.6 [0.38-0.82]	21.36 [0-60.35]
		10 -2	9.8 [4.1-15.4]	0.58 [0.08-1.15]	20.37 [0-85.01]
		10 -3	12.5 [4.8-20]	0.58 [0.42-0.74]	23.37 [6.36-39.57]
	5	10 -1	7.1 [0-13.9]	0.6 [0.19-1.06]	22.07 [0-58.57]
		10 -2	8.9 [4.7-13.2]	0.57 [0.43-0.74]	22.42 [1.9-41.84]
		10 -3	10.2 [4.8-16.2]	0.58 [0.46-0.71]	22.31 [5.48-41.46]
	Model averaging		8.6 [1.9-17.6]	0.6 [0.22-0.97]	22.71 [0-59.64]
	CI, confidence interval.

Timing of Treatment is Critical in SARS-CoV-2Gonçalves et al.

Conflict of Interest. J.G. has consulted for F. Hoffman-La Roche. All other authors declared no competing interests for this work.

Author Contributions. A.G., R.K., A.S.P., and J.G. wrote the manuscript. A.G., R.K., X.D.L., F.M., D.M., A.S.P., and J.G. designed the research. A.G., R.K., J.B., E.C., A.P., O.T., M.R.C., P.S., A.S.P., and J.G. performed the research. A.G., R.K., J.B., E.C., A.P., O.T., M.R.C., P.S., A.S.P., and J.G. analyzed the data.

Ethical Statement. Data were originally provided in Young et al. (https://doi.org/10.1001/jama.2020.3204) where "waiver of informed consent for collection of clinical data from infected individuals was granted by the Ministry of Health, Singapore" and "written informed consent was obtained from study participants."

Disclaimer. As Editor-in-Chief of CPT: Pharmacometrics & Systems Pharmacology, France Mentré was not involved in the review or decision process for this paper.


Bei Wang

Daniel Asarnow

Wen-Hsin Lee

Ching-Wen Huang

Bryan Faust

Patricia Miang

Lon Ng

Eve Zi

Xian Ngoh

Markus Bohn

David Bulkley

Andrés Pizzorno

Hwee Ching Tan

Chia Yin Lee

Rabiatul Adawiyah Minhat

Olivier Terrier

Mun Kuen Soh

Frannie Jiuyi Teo

Yvonne Yee

Chin Yeap

Yuanyu Hu

Shirley Gek

Kheng Seah

Sebastian Maurer-Stroh

Laurent Renia

Brendon John Hanson

Manuel Rosa- Calatrava

Aashish Manglik
email: aashish.manglik@ucsf.edu

Yifan Cheng
email: ycheng@ucsf.edu

Charles S Craik
email: charles.craik@ucsf.edu

Cheng-I Wang
email: wang_chengi@immunol.a-star.edu.sg

Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization

Keywords: SARS-CoV-2, COVID-19, SARS-CoV, coronavirus, spike protein, monoclonal antibody, receptor binding domain (RBD), phage display

In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection 1-4 . Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction 5 . Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

The ongoing COVID-19 pandemic, caused by a novel coronavirus SARS-CoV-2 originating in Wuhan, China, at the end of 2019, is one of the world's most devastating pandemics in living memory. As of July 1 2020, more than 10.7 million individuals have been infected worldwide resulting in nearly 518,000 deaths, and the numbers are still increasing. Currently only a single FDA-approved drug, Remdesivir, is available against COVID-19 and yet, it only shortened the duration of symptoms by 31% and had marginal improvement in survival, especially in the severely ill cases [START_REF] Beigel | Remdesivir for the Treatment of Covid-19 -Preliminary Report[END_REF] . In this context, additional and more effective prophylactic and therapeutic drugs are urgently needed.

The coronavirus spike polypeptide comprises two subunits, S1 and S2, and is responsible for mediating viral entry [START_REF] Tortorici | Structural insights into coronavirus entry[END_REF] . Upon binding to the host cell receptor through its S1 subunit, the spike protein undergoes dramatic conformational changes and proteolytic processing, triggering the S2 subunit to facilitate fusion of viral and cellular membranes. Both SARS-CoV-2 and SARS-CoV use the angiotensin converting enzyme 2 (ACE2) as the entry receptor to infect host cells 8- 10 . The receptor binding domain (RBD) located at the tip of the S1 subunit directly contacts ACE2 via the receptor binding motif (RBM), a small patch made up of about 20 amino acids [START_REF] Tai | Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine[END_REF][START_REF] Lan | Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor[END_REF] . Thus, a rational approach to the development of the anti-SARS-COV or anti-SARS-COV-2 therapy is to explicitly direct antibody discovery against the RBD/ACE2 interaction interface [START_REF] Zhou | Perspectives on therapeutic neutralizing antibodies against the Novel Coronavirus SARS-CoV-2[END_REF] .

Since the first report of COVID-19 outbreak, discovery of SARS-CoV-2 neutralizing monocloncal antibodies (NAbs) has undergone an unprecedent speed. Some existing potent SARS-CoV NAbs that target the RBD/ACE2 interaction were quickly tested for their crossreactivity against SARS-CoV-2 because of the high similarity between the two viruses [START_REF] Zhou | A pneumonia outbreak associated with a new coronavirus of probable bat origin[END_REF][START_REF] Wan | Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus[END_REF][START_REF] Yuan | A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV[END_REF] , yet only very few showed potent neutralization [START_REF] Yuan | A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV[END_REF][START_REF] Tian | Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody[END_REF][START_REF] Tai | Identification of SARS-CoV RBDtargeting monoclonal antibodies with cross-reactive or neutralizing activity against SARS-CoV-2[END_REF][START_REF] Pinto | Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody[END_REF] . Meanwhile, many SARS-CoV-2-specific NAbs have been discovered from immunized animals [START_REF] Wang | A human monoclonal antibody blocking SARS-CoV-2 infection[END_REF][START_REF] Wrapp | Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies[END_REF][START_REF] Hansen | Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail[END_REF] and COVID-19 convalescent patients [START_REF] Hansen | Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail[END_REF][START_REF] Ju | Human neutralizing antibodies elicited by SARS-CoV-2 infection[END_REF][START_REF] Chi | A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability[END_REF][START_REF] Brouwer | Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability[END_REF][START_REF] Wu | A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2[END_REF][START_REF] Rogers | Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model[END_REF][START_REF] Cao | Potent neutralizing antibodies against SARS-CoV-2 identified by highthroughput single-cell sequencing of convalescent patients' B cells[END_REF][START_REF] Shi | A human neutralizing antibody targets the receptor binding site of SARS-CoV-2[END_REF][START_REF] Wan | Human IgG neutralizing monoclonal antibodies block SARS-CoV-2 infection[END_REF][START_REF] Lou | Cross-neutralization antibodies against SARS-CoV-2 and RBD mutations from convalescent patient antibody libraries[END_REF] . Structural and biochemical studies indicated that most of these NAbs recognized epitopes within the ACE2 binding site, imposing direct competition between virus-ACE2 interaction [START_REF] Hansen | Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail[END_REF][START_REF] Ju | Human neutralizing antibodies elicited by SARS-CoV-2 infection[END_REF][START_REF] Brouwer | Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability[END_REF][START_REF] Wu | A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2[END_REF][START_REF] Cao | Potent neutralizing antibodies against SARS-CoV-2 identified by highthroughput single-cell sequencing of convalescent patients' B cells[END_REF][START_REF] Shi | A human neutralizing antibody targets the receptor binding site of SARS-CoV-2[END_REF] . Other antibodies were found to bind to epitopes that are outside the RBM [START_REF] Pinto | Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody[END_REF][START_REF] Wang | A human monoclonal antibody blocking SARS-CoV-2 infection[END_REF][START_REF] Hansen | Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail[END_REF] or even the RBD [START_REF] Chi | A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability[END_REF][START_REF] Brouwer | Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability[END_REF] , suggesting that various mechanisms of neutralization exist.

A number of these neutralizing antibodies have just entered clinical trials [START_REF] Hansen | Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail[END_REF] .

In addition to those derived from natural infection or immunization, potent neutralizing antibodies against pathogens can be isolated by in vitro selection from highly diverse combinatorial human libraries [START_REF] Vaughan | Human antibodies by design[END_REF][START_REF] Frenzel | Designing Human Antibodies by Phage Display[END_REF][START_REF] Lu | Development of therapeutic antibodies for the treatment of diseases[END_REF] . Interestingly, some of such antibodies often came from different antibody subtypes and recognized different epitopes [START_REF] Saphire | Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection[END_REF] . Following the release of the first SARS-CoV-2 genome, we set out to discover SARS CoV-2 neutralizing antibodies from a highly diverse human combinatorial antbody libray using a mechanism-based screening strategy. Here, we report the discovery of a potent NAb 5A6 with functional characterization in CHO-ACE2, Vero E6 cell line, and reconstituted human respiratory epithelium models, and reveal its molecular mechanism of action through cryogenic electron microscopy (cryo-EM).

Furthermore, the cross-neutralizing activity of 5A6 against major SARS-CoV-2 mutants is demonstrated, further highlighting its potential as a COVID-19 therapy.

Results and Discussions

Isolation and characterization of SARS-CoV-2 neutralizing Fabs from a naïve human

Fab phage display library

The naïve human Fab phage display library used in this project was constructed using the B cells donated by 22 healthy individuals, and has a diversity of 3 x 10 10 clones 32 . To isolate neutralizing antibodies, we directed antibody selection against the recombinant SARS-CoV-2 S protein RBD [START_REF] Wan | Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus[END_REF] , the domain responsible for viral attachment to the host receptor ACE2 as previously discussed. Following stringent in vitro selection conditions and only two rounds of biopanning, ELISA screening of 570 individual clones identified 243 Fabs that were able to bind to the SARS-CoV-2 spike RBD. While several anti-SARS-CoV-2 antibodies have been reported to neutralize the virus by unclear mechanisms [START_REF] Wang | A human monoclonal antibody blocking SARS-CoV-2 infection[END_REF][START_REF] Rogers | Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model[END_REF] , to be expeditious, we took a direct, mechanism-based approach by screening for those Fabs that block the RBD/ACE2 interaction in a competition ELISA. Fabs were incubated with RBD before transfer to ELISA plate wells coated with the recombinant ACE2 protein, and the extent of RBD/ACE2 blocking was determined by the presence of ACE2-bound RBD. Twenty-seven Fabs with unique sequences exhibiting various degrees of blocking were identified (Fig. 1a). Binding characteristics of these Fabs to the SARS-CoV-2 spike RBD protein are shown in Figure 1b. Of the 27 selected clones, 19 clones were reformatted into full length IgGs, expressed recombinantly in CHO cells, and their binding to the RBD confirmed by concentration-dependent ELISA (Extended Data Fig. 1). Six antibodies, clones 1F4, 2H4, 3D11, 3F11, 5A6 and 6F8, showed strong receptor blocking by bio-layer interferometry (BLI) (Fig. 1c) and in competition ELISA, with IC50 values ranging between 0.4 nM and 36.8 nM (Extended Data Fig. 2). All six completely blocked the RBD/ACE2 interaction at high concentrations, except clone 3D11 which plateaued at 50%. Two Fabs, 5A6 and 3D11 showed the highest affinities for RBD at 7.6 nM and 1.6 nM respectively, presumably because of their slower relative off rates (Fig. 1d, Extended Data Fig. 3). The Fabs had intrinsic affinity varying from 1.6 nM (3D11) to 120 nM (3F11). They also varied by two orders of magnitude in their association and dissociation rates with 3F11 having the fastest on-rate and 3D11 having the slowest off-rate. The IgG format showed strong, subnano-or even picomolar binding avidity to the SARS-CoV-2 spike RBD either by BLI (Fig. 1e, Extended Data Fig. 4) or by ELISA (Extended Data Fig. 5). Interestingly, only 3D11 was able to cross react with SARS-CoV spike RBD, although the binding was much weaker (Extended Data Fig. 5). The data suggest that 3D11 recognizes a distinct epitope that is different from the other five antibodies and is conserved between SARS-CoV-2 and SARS-CoV. Indeed, stepwise binding BLI assays confirmed that 5A6 and 3D11 have non-overlapping footprints on the RBD while 5A6 shares overlapping epitopes with the other four antibodies (Fig. 1f).

To evaluate the neutralization potential of these SARS-CoV-2 RBD reactive antibodies, we generated pseudovirus expressing SARS-CoV-2 or SARS-CoV spike glycoprotein tagged with a luciferase reporter, and established a pseudovirus neutralization assay featuring CHO cells stably expressing human ACE2 (CHO-ACE2) as viral targets. All six antibodies showed dose-dependent neutralization (Fig. 2a). 5A6 strongly neutralized the SARS-CoV-2 pseudovirus with an IC50 of 75.5 ng/ml, followed by 1F4 (108 ng/ml), 2H4 (109 ng/ml), 6F8 (428.3 ng/ml), and 3F11 (930.4 ng/ml). Interestingly, while 3D11 neutralized SARS-CoV-2 pseudovirus, the neutralization did not follow the typical sigmoidal dose response, suggesting a unique mechanism of inhibition. Consistent with our binding studies, none of these antibodies showed significant neutralization of the SARS-CoV pseudovirus, suggesting high specificity of our antibodies to SARS-CoV-2. Interestingly, although 3D11 was able to bind weakly to the SARS-CoV RBD (Extended Data Fig. 5), it was unable to significantly neutralize SARS-CoV pseudovirus (Fig. 2a). These six antibodies were further evaluated in Vero E6 cells for neutralizing activity against a clinical SARS-CoV-2 strain, isolated from a patient in Singapore [START_REF] Young | Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore[END_REF] . The 5 antibodies other than 3F11 showed >90% neutralization at the highest concentration (12.5 µg/ml), with 5A6 exhibiting an IC50 of 140.7 ng/ml, indicating >10-fold higher neutralizing potency than the other clones (Fig. 2b). In addition, 5A6 also potently neutralized a viral strain isolated in France with an IC50 of approximately 5 ng/ml (Fig. 2c).

The difference between patient isolates is of unclear origin as they share identical spike sequences, but may be due to intrinsic properties such as rate of replication controlled by other viral genes.

To explore the therapeutic potential of 5A6, we studied its neutralizing potency in physiologically relevant in vitro models. Human airway epithelia reconstituted using donorderived, primary differentiated nasal and bronchial cells (MucilAir TM HAE [START_REF] Pizzorno | Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia[END_REF] ) were infected with SARS-CoV-2 virus in the presence or absence of 5A6 or an equivalent isotype control IgG. Viral replication was quantified as the measured copy number of the viral genomes inside, and at the apical poles of, nasal and bronchial HAE. Figure 2d shows that viral replication was drastically reduced, being 10 3 to 10 4 -fold lower in the presence of 5A6 (75 or 150 ng/ml), while control IgG had little-to-no effect on infection. In addition, 5A6 also helped maintain epithelium integrity in the presence of virus, as evidenced by measurements of trans-epithelia electrical resistance. These results provided strong evidence in favour of the therapeutic potential of 5A6.

Mechanism of SARS-CoV-2 neutralization by 5A6

Given that viral neutralization IC50s are more similar in general to the binding avidity than to the intrinsic affinity, we hypothesized that 5A6 neutralized the virus via bivalent binding to spike proteins on the viral surface. To support this hypothesis, we compared the neutralization potencies of the monovalent Fab to the bivalent IgG. A pseudovirus neutralization assay showed that the monovalent 5A6 Fab (IC50=663.9 ng/m, Extended Data Fig. 6a) had a 10-fold reduction in the neutralization potency relative to the bivalent 5A6 IgG (IC50=75.5 ng/ml, Fig. 2a). Reduction of neutralizing potency was also observed for the other Fabs (Extended Data Fig. 6a). Furthermore, the neutralization potency of the Fabs was also drastically reduced or even diminished in a live virus neutralization assay (Extended Data Fig. 6b), consistent with results using pseudovirus. This suggested that 5A6 IgG neutralizes the SARS-CoV-2 virus via bivalent binding to the spike protein or by crosslinking multiple spike protein trimers. However, while IgGs 1F4, 2H4, 3D11, 3F11 and 6F8 have similar or higher avidity for immobilized RBD, 5A6 has much higher neutralizing potency against both pseudovirus and live SARS-CoV-2 viruses. We asked if the discrepancy could arise from the differences in structural arrangement between the artificially immobilized RBD proteins and the natural RBD displayed on the viral surface. To answer this question, we purified the SARS-CoV-2 pseudovirus by gradient centrifugation and immobilized the viral particles on the ELISA plates. Concentration-dependent binding curves revealed that 5A6 IgG was packed on the viral surface at a much higher density than the other four IgG antibodies or 5A6 Fab, as shown by the higher optical signal (Fig. 1g). This may be a result of a unique binding mode that allows 5A6 IgG to accommodate a denser structural arrangement on the viral surface. To this end, it has been well documented that effective viral neutralization requires antibody packing density exceeding a critical threshold [START_REF] Dowd | Antibody-mediated neutralization of flaviviruses: a reductionist view[END_REF][START_REF] Flamand | Mechanisms of rabies virus neutralization[END_REF][START_REF] Burton | A model for neutralization of viruses based on antibody coating of the virion surface[END_REF] , a criterion that was perhaps not easily achieved by the other five antibodies.

Structural basis of viral neutralization by antibody 5A6

To better understand the molecular mechanism of 5A6 bioactivity, we determined single-particle cryo-EM structures of the trimeric spike protein alone at 2.4 Å resolution (not shown) and in complex with 5A6 Fab at 2.4 Å (Fig. 3, Supplementary Movie 1, and Extended Data Fig. 7). In the uncomplexed spike protein structure, individual RBDs are seen adopting two major conformations, here termed "up" and "down." As previously reported, the resolution of an apo RBD in the up conformation is limited by its flexibility [START_REF] Walls | Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein[END_REF] . In the spike protein-5A6 Fab complex structure, the Fab hypervariable loops bind directly to surface loops at the tip of RBD. The RBD binding epitope of 5A6 (850 Å 2 ) overlaps the binding site of the ACE2 receptor suggesting direct, competitive inhibition of ACE2 binding and confirming the mechanism based biopanning strategy for identification of ACE2 blockers (Fig. 3b). 3D classification of the spike protein-5A6 Fab complex dataset reveals several different subclasses in which the Fab occupancies in the trimer varies with two hallmark features. One striking highlight is that 5A6 Fab can bind to RBD in both up and down conformations while engaging the same epitope (Fig. 3a-c). While the orientations of the three RBDs within the trimer are different, the atomic resolution model built from one 5A6-RBD interface is superimposable with the other 5A6-RBD structures, and no steric clashes result from modelling any configuration of the complex. The resolution of our structure at the interface between 5A6 Fab and RBD is sufficiently high (~2.7 Å local resolution) to allow accurate modelling of the 5A6/RBD interface (Fig. 3d). Four of the six complementary determining regions (CDRs) of 5A6, namely, loops H1, H2, H3 and L1 are involved in binding to RBD. A surfeit of tyrosines from H1 (Tyr 32), H2 (Tyr 53 and Tyr 59), L1 (Tyr 32), and L3 (Tyr 92), as well as Arg 98, Thr 101, Val 103 and Arg 104 in the H3 loop cooperate to stabilize the interface. These interactions provide a structural framework that suggests why 5A6 is not affected by the V483I mutation while Leu 94 in the L3 loop is a potential liability in binding to the 483A mutation.

The second hallmark feature is that two 5A6 Fabs are always found binding two neighbouring RBDs, one in an up position and one in a down position. A region of the light chain variable sequence of 5A6 that binds to the RBD in the down position forms a second interaction interface of 363 Å 2 with a neighbouring RBD in the up position (Fig. 3e). This interaction presumably contributes to locking the first RBD in the down conformation, and further stabilizes RBD in the up conformation, as it is better resolved than other RBDs in the up position with or without Fab bound. Notably, the constant domain of 5A6 bound to the down RBD unavoidably clashes with the model of ACE2 bound to the neighbouring up RBD (Fig. 3b left), perhaps further potentiating neutralization.

The distances between all Fabs bound to a single trimer are sufficiently close to allow two Fabs from a single IgG to bind simultaneously to two different RBDs within the trimer, and we reasoned that binding mode might explain the strong avidity and high surface density of 5A6 IgG discussed above. We therefore applied single-particle cryo-EM to the spike protein complexed with 5A6 IgG, and the resulting ~15 Å structure reveals that 5A6 IgG indeed binds to two RBDs from the same trimer (Fig. 3f). While the Fc domain of the IgG is not well resolved, due to conformational flexibility and the tendency of the IgG to induce spike aggregation, the density clearly indicates that two Fabs from the same IgG prefer binding to two neighbouring RBDs in up and down conformations, just as described above for free 5A6

Fab. Thus, one spike protein trimer can accommodate 1.5 IgGs with a single IgG binding two RBDs and the third RBD bound to another IgG, potentially bridging separate spike protein trimers.

Remarkably, 5A6 directly competes with ACE2, binds any configuration of trimer RBD conformations without steric hindrance from either the spike protein or other Fabs, permits binding of IgG with unaltered geometry, and may disrupt cooperative opening of the entire trimer by locking adjacent RBDs into up and down conformations.

Sensitivity of 5A6 and 3D11 to RBD mutations

A number of mutations resulting in amino acid changes in positions at or near the ACE2 interface have been isolated within more than 40,000 SARS-CoV-2 genomes sequenced to date. These include N439K in 246 samples (244 Scottish, 1 England, 1 Romania), V483A in 30 samples (26 USA/WA, 2 USA/UN, 1 USA/CT, 1 England), G476S in 18 samples (13 USA/WA, 2 USA/OR, 1 USA/ID, 1 USA/CT, 1 Belgium), S494P in 7 samples (3 USA/MI, 1 England, 1 Spain, 1 India, 1 Sweden), V483I in 2 English samples, and L455I together with F456V in one Brazilian sample. To determine if any of these mutant viruses could escape neutralization by 5A6 or 3D11, we produced the recombinant mutant RBD proteins to determine changes in binding avidity, and constructed mutant SARS-CoV-2 pseudoviruses to evaluate changes in neutralization potency. 5A6 IgG had a 4-fold reduction in binding avidity to the V483A mutation but was insensitive to the other five mutations (Fig. 4a, Extended Data Fig. 8a). Consistent with the binding results, 5A6 significantly lost neutralizing capacity against the V483A mutant pseudovirus. However, 5A6 largely retains activity against the other variants (Fig. 4c), including the D614G mutation that has spread at an alarming rate and become the dominant pandemic strain with a global frequency of 70.5% (GISAID), since its first identification in Europe in March [START_REF] Korber | Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2[END_REF] . Interestingly, 3D11 binding was not affected by any of these mutations (Fig. 4b, Extended Data Fig. 8b), but its neutralizing potency against the D614G mutant was partially reduced (Fig. 4d). The difference between the activity profiles of 5A6 and 3D11 could be explained by the different epitopes recognized by each antibody (Fig. 1f), and may argue for an antibody combination therapy to widen the neutralization spectrum against various mutant strains. While 3D11 has a weaker neutralizing potency than 5A6 in general, it can be further improved through antibody engineering, particularly via structureguided approaches once high-resolution structures of its spike protein complex are determined.

Hence, 3D11 could potentially rescue the loss of 5A6 neutralization potency in a virus strain bearing the V483A mutation, if these two antibodies are used in combination. Further structureaided engineering is underway to broaden the neutralizing spectrum of 5A6 against other mutations.

In summary, our study demonstrates that potent anti-SARS-CoV-2 neutralizing antibodies can be rapidly discovered within a highly diverse, naïve combinatorial human antibody library using a mechanism-based screening strategy. Antibody 5A6 neutralizes SARS-CoV-2 by simultaneously binding to two RBDs of different structural orientations in the spike trimer, and prevents ACE2 binding by direct competition. The high resolution structures of the 5A6/RBD complex reveal details of extensive tertiary interactions, and greater neutralization potency, including against viruses with variant RBDs, can likely be achieved through structure-guided engineering, paving the way for the development of effective therapeutics for COVID-19.

Fig. 1 .

 1 Fig. 1. Identification of anti-SARS-CoV-2 spike RBD antibodies that can block ACE2/RBD interactions. a, Blocking of ACE2/RBD (SARS-CoV-2) interactions by 27 Fab clones, tested by competition ELISA. b, Binding avidity of the 27 Fab clones to SARS-CoV-2 spike RBD protein by ELISA. c, Competition of RBD-binding between ACE2 and six IgGs by biolayer interferometry assay. A weak blocking IgG clone 1C3 was included as a negative control. d, Binding affinity and the rate of association and dissociation of 5 Fab clones to SARS-CoV-2 spike RBD protein by BLI. Values are the means  SD of two independent experiments. e, Binding avidity of six IgGs to the RBD by biolayer interferometry. The IgG concentration at 12.5 nM is shown here. f, Epitope binning of 5A6 by BLI analysis. Buffer alone, an isotype IgG and 5A6 IgG were included as controls. g, Binding of the IgG (solid lines, circles) and 5A6 Fab (dashed line, red triangle) to the purified SARS-CoV-2 pseudovirus. Data are presented as means  SD in duplicates and are representative of two independent experiments.

Fig. 2 .

 2 Fig. 2. Neutralization of the SARS-CoV-2 pseudovirus and live viruses by anti-SARS-CoV-2 spike RBD IgG antibodies. a, Infection of CHO-ACE2 cells by SARS-CoV-2 pseudovirus (left panel) and SARS-CoV pseudovirus (right panel) were determined in the presence of 1F4, 2H4, 3D11, 3F11, 5A6 and 6F8 recombinant IgGs. Luciferase activities in the CHO-ACE2 cells were measured, and the percent neutralization was calculated. Data are presented as means  SEM in triplicates and are representative of two independent experiments. b, Infection of Vero E6 C1008 cells by SARS-CoV-2 live virus (hCoV-19/Singapore/3/2020) were determined in the presence of 1F4, 2H4, 3D11, 3F11, 5A6 and 6F8

Fig. 3 .

 3 Fig. 3. Cryo EM structures. a, Cryo-EM density (left) and model (right) of 5A6 bound to spike protein in a 2:1 complex (2.4 Å global resolution). Spike monomers A, B, & C are shown in blue, green and red, respectively, with two 5A6 Fabs in goldenrod (bound to A, "down," and B, "up"). A second copy of the map is shown in transparent gray using a lower isovalue, to

Fig. 4 .

 4 Fig. 4. Sensitivity of 5A6 and 3D11 IgGs to RBD mutations. a, Binding avidity of IgGs 5A6 and b, to the wildtype RBD and six RBD mutants was determined by biolayer interferometry. IgG concentration at 12.5 nM is shown here. Neutralization of the SARS-CoV-2 pseudovirus with either wildtype (WT) or RBD mutations by c, 5A6 and d, 3D11 IgGs. Data are presented as means  SEM in triplicates and are representative of two independent experiments.

Acknowledgements

We thank Professor Yee-Joo Tan (Department of Microbiology, NUS; Institute of Molecular and Cell Biology, A*STAR) who kindly provided CHO-ACE2 cells and pXJ3'-S plasmid. We also thank Wong Pui San and Chye De Ho in the BSL 3 facility (DSO National Laboratories), as well as Dr. Pei Yin Lim (Singapore Immunology Network) for their technical assistance. We thank Professor Jason McLellan (University of Texas at Austin) for the prefusion S ectodomain. We thank Professor Joseph DeRisi and Professor Robert Fletterick (UCSF) and Dr. John Pak (CZI Biohub, San Francisco) for helpful discussions. This work was supported by core research grants provided to Singapore Immunology Network of the Biomedical Research Council (BMRC), the A*CCLERATE GAP fund (ACCL/19-GAP064-R20H-I) from the Agency of Science, Technology and Research (A*STAR, Singapore). Support was also provided by the National Institutes of Health Grant P50AI150476 (CSC, YC, MB), NIH Grants S10 S10OD020054 (YC) and S10OD021741 (YC) and the UCSF Program for Breakthrough Biomedical Research which is partially funded by the Sandler Foundation (C.S.C., Y.C., A.M.). Y.C. is an Investigator of the Howard Hughes Medical Institute.