The COVID-19 pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. Here, we characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting ~10-fold higher expression than its parental construct and the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

Broadly protective vaccines against known and pre-emergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics which may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days, and clearance of 0.22 mL/hr/kg, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study Day 6 following viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed at day 80 by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom and South Africa of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.One Sentence SummaryThree mutations allowed SARS-CoV-2 to evade the polyclonal antibody response of a highly neutralizing COVID-19 convalescent plasma.

1The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated 2 efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health 3 emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion 4 protein that is metastable and difficult to produce recombinantly in large quantities. Here, we 5 designed and expressed over 100 structure-guided spike variants based upon a previously 6 determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical 7 and structural characterization of these variants identified numerous individual substitutions that 8 increased protein yields and stability. The best variant, HexaPro, has six beneficial proline 9 substitutions leading to ~10-fold higher expression than its parental construct and is able to 10 withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution 11 cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-12 yield production of a stabilized prefusion spike protein will accelerate the development of 13 vaccines and serological diagnostics for SARS-CoV-2. 14 3 INTRODUCTION 15 Coronaviruses are enveloped viruses containing positive-sense RNA genomes. Four human 16 coronaviruses generally cause mild respiratory illness and circulate annually. However, SARS-17 CoV and MERS-CoV were acquired by humans via zoonotic transmission and caused outbreaks 18 of severe respiratory infections with high case-fatality rates in 2002 and 2012, respectively 1,2 . 19 SARS-CoV-2 is a novel betacoronavirus that emerged in Wuhan, China in December 2019 and 20 is the causative agent of the ongoing COVID-19 pandemic 3,4 . As of May 26, 2020, the WHO has 21 reported over 5 million cases and 350,000 deaths worldwide. Effective vaccines, therapeutic 22 antibodies and small-molecule inhibitors are urgently needed, and the development of these 23 interventions is proceeding rapidly. 24 Coronavirus virions are decorated with a spike (S) glycoprotein that binds to host-cell 25 receptors and mediates cell entry via fusion of the host and viral membranes 5 . S proteins are 26 trimeric class I fusion proteins that are expressed as a single polypeptide that is subsequently 27cleaved into S1 and S2 subunits by cellular proteases 6,7 . The S1 subunit contains the receptor-28 binding domain (RBD), which, in the case of SARS-CoV-2, recognizes the angiotensin-29 converting enzyme 2 (ACE2) receptor on the host-cell surface [8][9][10] . The S2 subunit mediates 30 membrane fusion and contains an additional protease cleavage site, referred to as S2′, that is 31 adjacent to a hydrophobic fusion peptide. Binding of the RBD to ACE2 triggers S1 dissociation, 32 allowing for a large rearrangement of S2 as it transitions from a metastable prefusion 33 conformation to a highly stable postfusion conformation 6,11 . During this rearrangement, the 34 fusion peptide is inserted into the host-cell membrane after cleavage at S2′, and two h...

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)–directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of “public” antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that “public” NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection.