key: cord-0969267-oil8qslm
authors: Piepenbrink, Michael S.; Park, Jun-Gyu; Desphande, Ashlesha; Loos, Andreas; Ye, Chengjin; Basu, Madhubanti; Sarkar, Sanghita; Chauvin, David; Woo, Jennifer; Lovalenti, Philip; Erdmann, Nathaniel B.; Goepfert, Paul A.; Truong, Vu L.; Bowen, Richard A.; Walter, Mark R.; Martinez-Sobrido, Luis; Kobie, James J.
title: Potent universal-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody
date: 2022-03-07
journal: bioRxiv
DOI: 10.1101/2022.03.05.483133
sha: 3b7c2772b0474d2e2af688ea2eaa6df487ee96ab
doc_id: 969267
cord_uid: oil8qslm

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel β-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human β-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human β-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

SARS-CoV-2 is the third known emergence of a coronavirus (CoV) with the ability to cause substantial human morbidity and mortality. The SARS-CoV-2 pandemic has resulted in over 5 million deaths worldwide in two years and painfully highlights the vulnerability of humanity to novel CoV. Despite the rapid development of vaccines exhibiting high levels of efficacy and low levels of side effects, global vaccine implementation has been slow. As a result, SARS-CoV-2 virus has infected many human hosts, allowing for the evolution of new variants that have the potential to evade the immune response elicited by previous infection or vaccination.

Although first generation SARS-CoV-2 vaccines have been highly effective at preventing severe disease, including from VoC, the humoral immunity induced by vaccination and natural infection is overwhelmingly dependent on a neutralizing antibody response targeted to the Receptor Binding Domain (RBD) of the Spike (S) glycoprotein. The RBD, which mediates the initial attachment of SARS-CoV-2 to its primary receptor, angiotensin converting enzyme 2 (ACE2), and the S1 domain overall have undergone substantial antigenic diversity since the initial emergence of SARS-CoV-2. Mutations within RBD and S1 dramatically negate the neutralizing activity of plasma antibodies (Abs) against SARS-CoV-2 VoC that are generated from vaccinated or infected individuals and enhance the transmissibility and pathogenicity of VoC (1) (2) (3) (4) .

A good example of significant immune escape is the emergence of the Omicron VoC, with numerous mutations in the RBD that contribute to reduced neutralization by therapeutic human monoclonal antibodies (hmAbs) which are against RBD, and by vaccine and infection -induced plasma Abs (5, 6) .

The Spike is assembled as a homotrimer with ~24 molecules located on the surface of each SARS-CoV-2 virion (7) . While being synthesized, the S protein is initially cleaved by furin or furin-like proprotein convertase in the Golgi resulting in the S1 domain being non-covalently linked to the S2 domain (i.e. stalk) of the protein (8) . Mature viruses are released from infected cells after virus containing vesicles fuse with the cell membrane.

CoV S is a class I virus fusion protein (9) , with the S1 domain mediating attachment primarily through its RBD, while the S2 domain mediates fusion to the host cell membrane and entry. The SARS-CoV-2 viral fusion mechanism is conserved across numerous pathogen proteins including HIV envelope, influenza hemagglutinin, Ebola GP, and RSV fusion protein (10) . While CoV S1 domains exhibit substantial variation to allow recognition of different host receptors, CoV S2 domains are highly conserved overall (90% between SARS-CoV and SARS-CoV-2), which is consistent with their conserved function. For membrane fusion to occur a highly dynamic transformation must occur, requiring precise interactions of various S2 subdomains (11) , suggesting there is low tolerance for S2 variation without compromising viral fitness. Antibody responses to S2 are increased in some individuals following SARS-CoV-2 infection and plasma S2 Ab response are associated with survival after coronavirus disease 2019 infection (12) . However, the development of S2-specific Abs following SARS-CoV-2 natural infection or standard vaccination is limited compared to those specific for S1 (13, 14) . While most S1 Abs are neutralizing, only a small fraction of S2

Abs are neutralizing (13) .

Many SARS-CoV-2 neutralizing antibodies also target the N-terminal domain (NTD).

While neutralizing Abs to NTE have been identified, new SARS-CoV-2 variants such as Omicron, have gained several mutations in the NTD and RBD of the Spike protein that promote immune evasion. With the substantial number of people unvaccinated and immunocompromised, and emergence of new variants that can evade immune protection, a great need remains for new therapeutics that neutralize SARS-CoV-2, despite its continued evolution. Furthermore, there is a potential treatment advantage of combining Abs that inhibit both ACE2 binding and viral fusion.

Therapeutic mAbs are typically delivered systemically through intravenous infusion or intramuscular injection, which is highly inefficient and slow in achieving optimal concentrations in the respiratory tract for the treatment of respiratory infections (15) , including SARS-CoV-2 (16) . We have previously demonstrated that direct respiratory delivery of a SARS-CoV-2 RBD-specific hmAb enables substantial dose-sparing therapeutic activity in hamsters and here evaluate this delivery mechanism for the treatment of SARS-CoV-2 and SARS-CoV with a S2-specific hmAb.

To more precisely identify the potential of the SARS-CoV-2 S2 specific B cell response, a panel of S2-specific hmAbs were isolated and their molecular features, reactivity profiles, and in vitro and in vivo antiviral activities were defined. Several of these hmAbs demonstrate broad CoV reactivity, neutralizing, and antibody-dependent phagocytosis activity. With the most potent and broad S2-specific hmAb, 1249A8 exhibiting prophylactic and therapeutic activity against SARS-CoV-2 and SARS-CoV in multiple animal models, demonstrating the universal CoV therapeutic potential of S2 hmAb directly. The combination of 1249A8 with and S1-speciific hmAb 1213H7 was evaluated for possible synergistic effects and delivered to the respiratory tract for improved dose delivery efficiency.

To identify SARS-CoV-2 S2specific human B cells, two complementary recombinant proteins were designed and produced; a pre-fusion state stabilized SARS-CoV-2 (S2-STBL) and a SARS-CoV/SARS-CoV-2 full Spike chimera consisting of SARS-CoV S1 and SARS-CoV-2 S2 (SARS-CoV-1/2 S1S2) ( Figure 1A) . Initial testing of plasma from COVID-19 convalescent patients was performed to identify those with high avidity IgG binding titers against S2 (Figure 1B) . Using fluorescent S2-STBL and S1S2 chimera tetramers, peripheral blood memory B cells from several subjects were single-cell sorted by flow cytometry ( Figure 1C ) and recombinant fully human IgG1 mAbs (hmAbs) were generated. Seventeen hmAbs with reactivity to SARS-CoV-2 S2 protein resulted ( Figure 1D ). In general, most hmAbs bound commercial preparations of SARS-CoV-2 S, as well as S2-STBL and SARS-CoV-1/2, as shown in the plasma profiling. Binding to S2-STBL and SARS-CoV-1/2 S1S2 was more discriminating, as also evident in the plasma profiling. The previously reported S2-specific hmAb CC40.8 (17) was included as a positive control. Off-target binding to SARS-CoV-2 S1 was not evident.

phagocytosis activity. The functional activity of S2 hmAbs against SARS-CoV-2 was tested. The hmAbs that showed the greatest binding to at least one S2 protein by ELISA were tested by live virus and pseudovirus-based neutralization assays. Several hmAbs did not show neutralization capacity, even at the highest concentration (50 μg/ml) ( Figure 2A+B ). Eight hmAbs demonstrated neutralization of SARS-CoV-2 D614G pseudovirus (PsV) and were tested further, of which four hmAbs (1249A8, 1242C6, 1250D2, and 1235C10) effectively neutralized both PsV and live virus including Beta and Delta VoC. 1249A8 emerged as having the broadest and most potent neutralizing activity, with comparable NT 50 (neutralization titer at 50% inhibition) to CV3-25, a previously described S2 neutralizing hmAb (18) , with the notable exception of SARS-CoV-2 Beta which was not neutralized by CV3-25.

The Fc effector function of the S2 hmAbs was assessed by antibody dependent cellular phagocytosis (ADCP) of SARS-CoV-2 Wuhan-Hu-1 Spike coated beads ( Figure 2C ).

1242F4 and 1250E10 had the highest ADCP activity, similar to the previously described S2-specific hmAb S2P6 (19) . Both 1246C2 and 1246H7 had activity that was only slightly higher than the isotype control indicating very little Fc effector function, and consistent with their limited binding and neutralizing activity. 1249A8 had 4.2 fold greater ADCP activity than the isotype control which was similar to the S2 hmAbs CC40.8 and CV3-25. The SARS-CoV-2 RBD specific mAb 1213H7 (16, 20, 21) , had the greatest (~8 times greater than isotype) ADCP activity. Together these results suggest S2 hmAbs have the potential to eliminate SARS-CoV-2 through both neutralization and Fc-dependent effector functions. Four S2 protein fragments (S2 Frag1-Frag4) that cover different regions of the S2 amino acid sequence were produced to identify the binding epitope of 1249A8 ( Figure 2D ). S2 fragment binding assays localized the 1249A8 binding epitope to S2 residues 1131-1171 (S2-Frag4), which contains the conserved stem helix region (residues 1148-1158) of S2, previously reported to be recognized by mAbs CV3-25 (18), CC40.8 (17) , and S2P6 (19) .

Molecular characteristics of S2 hmAbs. The most potent neutralizing hmAb, 1249A8 was isolated from an IgG1 expressing B cell and exhibited substantial somatic hypermutation including 16.7% amino acid mutation from germline in the heavy chain variable region, and 7.6% amino acid mutation from germline in the light chain variable region ( Table 1) . The 1249A8 hmAb is member of the same clonal lineage that includes (21) . Mice were also treated alone or in combination with a modest dose of 1213H7 (5 mg/kg), a broad and potent SARS-CoV-2 RBD specific hmAb we have previously described (16, 20, 21) . All mice treated with the isotype control hmAb had declining body weight following infection that required euthanasia by D9 ( Figure   3A+B ). Mice treated with 10 mg/kg 1249A8 showed a milder weight loss with 60% of the mice surviving. Mice treated with 40 mg/kg of 1249A8 prior to infection, as well as those treated with 1213H7 or the combination of both did not have weight loss and all survived. Both 10 mg/kg and 40 mg/kg 1249A8 significantly (p<0.05) reduced nasal virus at day (D) 2, with 1249A8 and 1213H7 combination treated mice not having detectable virus, with all 1249A8 treated mice having viral titer below the limit of detection at D4 (Figure 3C) , with viral burden dominated by rSARS-CoV-2 Beta/mCherry, as previously described (21) . Lungs of mice that were treated with the isotype control mAb showed intense fluorescent radiance for both rSARS-CoV-2 WA-1/Venus and rSARS-CoV-2 Beta/mCherry in left and right hemispheres by D2 following infection and markedly increased at D4, and minimally visually evident in the 1249A8, 1213H7, and combination treated mice ( Figure 3D) . Lung viral titer was reduced in mice treated with either 1249A8 doses by ~2 log at D2, and to below detection limit at D4 compared to isotype control hmAb treated mice ( Figure 3E ). The reduction in viral burden by 1249A8 was for both rSARS-CoV2 WA-1/Venus and rSARS-CoV2

Beta/mCherry (Figure 3E , F, G) and was consistent with significant (p<0.05) reduction in lung pathology ( Figure 3H ). These results indicate that 1249A8 alone and in combination with the RBD mAb 1213H7 can broadly limit SARS-CoV-2 upper and lower respiratory viral burden and lung pathology, including the original lineage A and the lineage B Beta VoC. The breadth of the S2 binding activity of 1249A8 was further evaluated by avidity ELISA in the presence of urea, confirming its binding to SARS-CoV and MERS-CoV Spike, and also demonstrating its binding to OC43 and HKU-1 seasonal β -CoV ( Figure 4B) . No binding to the seasonal α-CoV 229E or NL63 Spike proteins was detected (not shown).

1249A8 uniquely had substantial broad binding to recombinant S proteins from MERS-CoV, OC43, and HKU1 compared to CV3-25 and CC40.8 which did not recognize MERS-CoV S, and S2P6 which had lower reactivity to HKU-1 S compared to 1249A8 Spikes as compared to S2P6, as a result of a S2P6 having a faster off-rate (kd) ( Figure   4C ). 1249A8 effectively neutralizes both live SARS-CoV (NT 50 =570 ng/ml) and MERS-CoV (NT 50 =5,830 ng/ml) ( Figure 4D ). As expected based on binding activity, CV3-25 did not neutralize MERS-CoV. Additionally, 1249A8 has ADCP activity against MERS-CoV Spike coated beads ( Figure 4E) . These results indicate that several SARS-CoV-2 S2 specific hmAbs have broad beta-CoV reactivity, with 1249A8 demonstrating universal β -CoV functional activity.

CoV-2 Omicron. The emergence of the SARS-CoV-2 Omicron VoC and its substantial evasion of neutralizing antibodies (2, 5, 6) necessitated testing of 1249A8. 1249A8 retains high affinity (KD = 0.52 nM) for the SARS-CoV-2 Omicron Spike ( Figure 5A) and neutralizing activity (NT 50 = 2407 ng/ml) against live SARS-CoV-2 Omicron virus ( Figure 5B ). We also observed potent SARS-CoV-2 Omicron neutralization of 1213H7 (NT 50 = 64 ng/ml). As clinical development of an S2 mAb would likely include a RBD specific mAb, and as these mAbs target distinct Spike domains (S1 and S2) and steps in the infection process (attachment and fusion) we sought to determine their combinatorial activity. In the presence of 50 ng/ml 1213H7, the NT 50 of 1249A8 against SARS-CoV-2 Omicron was reduced to 1338 ng/ml, and complementarily, in the presence of 2000 ng/ml of 1249A8 the NT 50 of 1213H7 was reduced to 26 ng/ml, with similar effect observed for SARS-CoV-2 WA-1 (not shown), suggesting co-operative activity of the S2 and RBD mAbs in neutralizing SARS-CoV-2. Treatment of K18 hACE2 mice with 1249A8 alone i.p. prior to challenge with 10 5 plaque forming units (PFU) of SARS-CoV-2 Omicron significantly reduced upper and lower respiratory viral burden compared to isotype control treated mice ( Figure 5C+D ). Treatment with 1213H7 alone i.p. also significantly reduced upper and lower respiratory viral burden. The combination of 1249A8 and 1213H7 significantly reduced viral burden, being more pronounced when the hmAbs were administered directly to the respiratory tract through intranasal (i.n.) delivery, with 50% of the mice not having detectable virus in the nasal turbinate at D2 and D4, and lungs at day 2. None of the mice treated i.n. with the 1249A8 and 1213H7 combination had detectable virus in the lungs at D4. This was consistent with the significant reduction in lung pathology in these mice ( Figure 5E ). These results indicate that direct respiratory administration of the RBD and S2 mAb cocktail of 1213H7 and 1249A8, respectively significantly reduce SARS-CoV-2 Omicron viral burden through a cooperative effect.

β -coronavirus therapeutic activity.

Given the broad β -CoV activity of 1249A8 in vitro and its demonstrated prophylactic activity against SARS-CoV-2 WA-1, Beta, and Omicron in K18 hACE2 mice, we evaluated its therapeutic potential in hamsters when delivered directly to the respiratory tract. Hamsters were infected with SARS-CoV-2 Delta and 12 h p.i. were treated with a single dose of hmAb delivered intranasally. Isotype control hmAb treated and untreated hamsters exhibited ~15% body weight loss within 6 days post infection (d p.i.), with minimal weight loss in hamsters treated with the 1249A8 or 1213H7 alone, or in combination ( Figure 6A ). Combined treatment with 8 mg/kg of 1249A8 and 2 mg/kg 1213H7 significantly reduced upper respiratory viral burden by ~3 logs ( Figure 6B ) and lower respiratory viral burden by ~6 logs (Figure 6C+D ) compared to control groups.

To assess pan β -CoV therapeutic activity, hamsters were infected with SARS-CoV, Urbani strain, and 12 h p.i. treated similarly with a single dose of hmAb delivered intranasally. As expected, untreated hamsters and those treated isotype control hmAb lost 15 to 20% of body weight by 7 d p.i.. Hamsters treated with 2, 4 or 8 mg/kg of 1249A8 had <5% weight loss, and those treated with 8 mg/kg 1249A8 alone or in combination with 2 mg/kg of 1213H7 actually gained weight by 7 d p.i. (Figure 7A) . A significant reduction in upper respiratory viral burden between D1-D3 as determined by oropharyngeal swabbing was evident in all hmAb treated groups ( Figure 7B ). At D3 significant reduction in lung viral titer was evident in hamsters treated with 1249A8 alone and in combination with 1213H7 ( Figure 7DE ). These results indicate the S2 hmAb 1249A8 has broad β -CoV in vivo therapeutic activity. In SARS-CoV-2 infection, the RBD is immunodominant, having several antigenic sites that account for 90% of the neutralizing activity of convalescent plasma (22) .

Unfortunately, β -CoV cross-reactive Spike S1/RBD neutralizing antibodies are rare and most S1/RBD mAbs have a very narrow specificity. The Spike protein S2 domain is more highly conserved across β -CoV, and here we have identified several hmAbs from convalescent patients that are highly cross reactive for all variants of SARS-CoV-2 tested. Further, four of these hmAbs also showed cross reactivity to SARS-CoV and MERS-CoV, with 1249A8 hmAb also effectively neutralizing SARS-CoV and MERS-

A few S2-specific hmAbs with broad activity have been reported in the literature, and we have included them as possible for comparison in our in vitro assessments of 1249A8.

1249A8 is distinct from CV3-25 in its ability to neutralize SARS-CoV-2 Beta VoC, more potent neutralization of SARS-CoV-2 WA-1 consistent with its more potent protection from weight loss and death in K18 hACE2 mice (23) , increased binding to OC43 and HKU-1 S, and its ability to bind and neutralize MERS-CoV (24) . 1249A8 is distinct from S2P6 in its higher affinity for SARS-CoV-2 S and MERS-CoV S, increased binding to HKU-1 S2, and ability to significantly prevent viral burden from SARS-CoV-2 Beta VoC infection (19) . 1249A8 is distinct from CC40.8 in its ability to bind MERS-CoV S (17) .

Together these findings suggest that 1249A8 is a uniquely broad and potent S2 hmAb that universally recognizes all human β -CoV. 1213H7 demonstrated remarkable breadth for an RBD-specific hmAb in its ability to neutralize all SARS-CoV-2 variants tested including recent Omicron VoC, which evaded many of the hmAbs that were being used clinically for treating . This suggests that 1213H7 is well suited for the S1 targeting component of an S1/S2 hmAb cocktail for the treatment and prevention of SARS-CoV-2.

Our results suggest that 1249A8 recognizes a yet undefined epitope within stemhelix/HR2 region of S2, and crystallography efforts to precise determine the epitope are ongoing. The ability of 1249A8 to directly neutralize (19) found that the hybrid mAb reduced replicating viral titers to a greater degree than the human counterpart. This context suggests that the Fceffector function of 1249A8 may contribute to in vivo viral clearance and warrants definitive assessment.

The substantial somatic hypermutation that is observed in 1249A8 including 16.7% VH and 13.5% Vk amino acid mutation from germline is higher than would be expected after a primary viral infection. And given its cross-reactivity with seasonal CoV OC43 and HKU-1 suggests that 1249A8 arose from a pre-existing memory B cell that was CoV utilize a variety of host receptors to gain entry, infect, and disseminate. The seasonal CoV including β-CoV OC43 and HKU1 infect through S1 binding to ubiquitous 9-O-acetyl sialic acid residues on host glycoproteins and lipids, α-CoV 229E utilizes S1 binding to aminopeptidase N (CD13), and α-CoV NL63 entry using both ACE2 and heparin sulfate proteoglycans; and β-CoV MERS-CoV uses dipeptidyl peptidase 4 (DPP4). Amongst the CoV, including β-CoV OC43 and HKU1, the precise binding sites for attachment receptors has still not been conclusively defined(26-28), suggesting possible heterogeneity or fluidity in the evolution of CoV as they adapt to humans.

Although SARS-CoV-2 RBD binds with high affinity to ACE2, facilitating attachment to host cell and ultimate infection, and to date SARS-CoV-2 entry and pathology appears highly dependent on ACE2, some reports have described ACE2-independent SARS- Monoclonal antibody production. Single B cells were sorted using a FACSMelody (BD Biosciences) into 96-well PCR plates containing 4 µl of lysis buffer as previously described (35) . Plates were immediately frozen at −80°C after sorting until thawed for reverse transcription and nested PCR performed for IgH, Igλ, and Igκ variable gene transcripts as previously described (35, 36) . Paired heavy and light chain genes were cloned into IgG1 expression vectors and were transfected into HEK293T cells and culture supernatant was concentrated using 100,000 MWCO Amicon Ultra centrifugal filters (Millipore-Sigma, Cork, Ireland), and IgG captured and eluted from Magne Protein A beads (Promega, Madison, WI) as previously described (35, 36) . Immunoglobulin sequences were analyzed by IgBlast (www.ncbi.nlm.nih.gov/igblast) and IMGT/V-QUEST (http://www.imgt.org/IMGT_vquest/vquest) to determine which sequences should lead to productive immunoglobulin, to identify the germline V(D)J gene segments with the highest identity, and to scrutinize sequence properties. CV3-25, S2P6, and CC40.8 were previously described (17) (18) (19) CoV neutralization. hmAbs were tested for neutralization of live SARS-CoV-2, SARS-CoV, and MERS-CoV as previously described (39) . Vero E6 cells (96-well plate format, 4 × 10 4 cells/well, quadruplicate) were infected with 100-200 PFU/well of SARS-CoV-2.

SARS-CoV-2 Omicron neutralization was performed in Vero AT using 600 PFU/well. Fluorescent images of lungs were photographed using an IVIS (AMI HTX), and the brightfield images of lungs were taken using an iPhone 6s (Apple). Nasal turbinate and lungs from mock or infected animals were homogenized in 1 mL of PBS for 20 s at 7,000 rpm using a Precellys tissue homogenizer (Bertin Instruments). Tissue homogenates were centrifuged at 12,000 × g (4 °C) for 5 min, and supernatants were collected and titrated by plaque assay and immunostaining as previously described. Statistical analysis. Significance was determined using GraphPad Prism, v8.0. Twotailed t-test were applied for evaluation of the results between treatments. p<0.05 was considered significant. For statistical analysis viral titers were log transformed and undetectable virus was set to the limit of detection.

We are grateful for the clinical research staff that enabled this project and for the technical assistance provided by Christopher Bates, the technical guidance provided by Justin Roth, the virology expertise provided by Ilya Frolov, and the assistance of the 

All relevant data are within the manuscript. were infected with 600 pfu SARS-CoV-2 Omicron (BEIR) and after 1 h of viral adsorption, the indicated mAb(s) was added and at 24 h.p.i infected cells were fixed for virus titration by immunostaining assay. 1213H7 and 1249A8 were tested alone (open symbols) and together keeping 1213H7 constant (50 ng/ml) or 1249A8 constant (2 μ g/ml) and titrating the reciprocal mAb (closed symbols). Resulting NT 50 (ng/ml) are indicated. K18 hACE2 mice were treated with 1249A8 (40 mg/kg), 1213H7 (10 mg/kg), or isotype control mAb (40 mg/kg) either alone or in combination i.p. or i.n. as indicated and 24 h later challenged i.n. with 10 5 PFU SARS-CoV-2 Omicron (BEIR) and virus titer in nasal turbinates (C) and lungs (D) determined at 2 and 4 dpi by plaque assay and gross lung pathology measured (E). Each symbol represents and individual animal.

Dotted line indicates limit of detection. *indicates p<0.05 compared to isotype control group as determined by t test. (B) Oropharyngeal swabs were collected days 1, 2, and 3 p.i. and sum of virus titer indicated. Nasal turbinate (C), cranial lung (C), and caudal lung (E) viral titers were measured at 3 d p.i. by plaque assay. Each symbol represents an individual animal. *indicates p<0.05 compared to isotype control group as determined by t test.

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