Murine Monoclonal Antibodies against the Receptor Binding Domain of SARS-CoV-2 Neutralize Authentic Wild-Type SARS-CoV-2 as Well as B.1.1.7 and B.1.351 Viruses and Protect In Vivo in a Mouse Model in a Neutralization-Dependent Manner.
TL;DR: In this article, mouse monoclonal antibodies against different epitopes on the receptor binding domain (RBD) and assessed binding and neutralization of authentic SARS-CoV-2.
Abstract: After first emerging in late 2019 in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized, but the supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the full-length spike protein of SARS-CoV-2. In this study, we generated mouse monoclonal antibodies (MAbs) against different epitopes on the RBD and assessed binding and neutralization of authentic SARS-CoV-2. We demonstrate that antibodies with neutralizing activity, but not nonneutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the MAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variant in vitro. IMPORTANCE Cross-neutralization of SARS-CoV-2 variants by RBD-targeting antibodies is still not well understood, and very little is known about the potential protective effect of nonneutralizing antibodies in vivo. Using a panel of mouse monoclonal antibodies, we investigate both of these points.
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01 Mar 2022
TL;DR: In this paper , the S:655Y mutation in SARS-CoV-2 enhances spike protein cleavage and fusogenicity and was able to outcompete S: 655H in the hamster model and in a human primary airway system.
Abstract: •The S:655Y mutation in SARS-CoV-2 enhances spike protein cleavage and fusogenicity•The S:655Y mutation increases transmissibility in vivo•S:655Y was able to outcompete ancestral S:655H in vivo•SARS-CoV-2 VOCs evolve to acquire an increased spike cleavage and fusogenic ability SARS-CoV-2 lineages have diverged into highly prevalent variants termed “variants of concern” (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission. SARS-CoV-2 lineages have diverged into highly prevalent variants termed “variants of concern” (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission. SARS-CoV-2 has been spreading worldwide and causing millions of infections and deaths since its emergence in Wuhan, China in late 2019. Apart from humans, ferrets, cats, dogs, Syrian golden hamsters, and nonhuman primates are also susceptible to SARS-CoV-2 infection and transmission (Fenollar et al., 2021Fenollar F. Mediannikov O. Maurin M. Devaux C. Colson P. Levasseur A. Fournier P.E. Raoult D. Mink, SARS-CoV-2, and the Human-Animal Interface.Front. Microbiol. 2021; 12: 663815Crossref PubMed Scopus (39) Google Scholar; Shi et al., 2020Shi J. Wen Z. Zhong G. Yang H. Wang C. Huang B. Liu R. He X. Shuai L. Sun Z. et al.Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.Science. 2020; 368: 1016-1020Crossref PubMed Scopus (873) Google Scholar). In addition, cases of viral spread in mink farms and mink-to-human cross-species transmission have been reported (Hammer et al., 2021Hammer A.S. Quaade M.L. Rasmussen T.B. Fonager J. Rasmussen M. Mundbjerg K. Lohse L. Strandbygaard B. Jørgensen C.S. Alfaro-Núñez A. et al.SARS-CoV-2 Transmission between Mink (Neovison vison) and Humans, Denmark.Emerg. Infect. Dis. 2021; 27: 547-551Crossref PubMed Scopus (91) Google Scholar; Oude Munnink et al., 2021Oude Munnink B.B. Sikkema R.S. Nieuwenhuijse D.F. Molenaar R.J. Munger E. Molenkamp R. van der Spek A. Tolsma P. Rietveld A. Brouwer M. et al.Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans.Science. 2021; 371: 172-177Crossref PubMed Scopus (376) Google Scholar). The spike (S) glycoprotein of SARS-CoV-2 is the main determinant of host tropism and susceptibility and the main target of antibody responses (Tong et al., 2021Tong P. Gautam A. Windsor I. Travers M. Chen Y. Garcia N. Whiteman N.B. McKay L.G.A. Lelis F.J.N. Habibi S. et al.Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.Preprint at bioRxiv. 2021; https://doi.org/10.1101/2021.03.10.434840Crossref Scopus (0) Google Scholar). Therefore, the emergence of adaptive mutations present in the S protein can strongly affect host tropism and viral transmission (Johnson et al., 2020Johnson B.A. Xie X. Kalveram B. Lokugamage K.G. Muruato A. Zou J. Zhang X. Juelich T. Smith J.K. Zhang L. et al.Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis.Preprint at bioRxiv. 2020; https://doi.org/10.1101/2020.08.26.268854Crossref Scopus (0) Google Scholar; Starr et al., 2020Starr T.N. Greaney A.J. Hilton S.K. Ellis D. Crawford K.H.D. Dingens A.S. Navarro M.J. Bowen J.E. Tortorici M.A. Walls A.C. et al.Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.Cell. 2020; 182: 1295-1310.e20Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). The S protein is composed of 2 subunits: S1, which contains the receptor-binding domain (RBD) that initiates infection by binding to the angiotensin converting enzyme 2 (ACE2) receptor present in the host cell surface, and the S2 subunit that mediates fusion between viral and cellular membranes (Duan et al., 2020Duan L. Zheng Q. Zhang H. Niu Y. Lou Y. Wang H. The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens.Front. Immunol. 2020; 11: 576622Crossref PubMed Scopus (98) Google Scholar; Huang et al., 2020Huang Y. Yang C. Xu X.F. Xu W. Liu S.W. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19.Acta Pharmacol. Sin. 2020; 41: 1141-1149Crossref PubMed Scopus (602) Google Scholar). To fuse with the host cell, the S protein needs to be cleaved by cellular proteases at the S1/S2 and S2′ sites. Importantly, the S1/S2 site of SARS-CoV-2 viruses contains a multibasic furin motif (681PRRXR685) absent in other beta coronaviruses (Coutard et al., 2020Coutard B. Valle C. de Lamballerie X. Canard B. Seidah N.G. Decroly E. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade.Antiviral Res. 2020; 176: 104742Crossref PubMed Scopus (885) Google Scholar; Hoffmann et al., 2020Hoffmann M. Kleine-Weber H. Pohlmann S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells.Mol. Cell. 2020; 78: 779-784.e5Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar) that can be processed by furin proteases but also by transmembrane serine proteases, such as TMPRSS2, or by cathepsins present in the endosomes (Bestle et al., 2020Bestle D. Heindl M.R. Limburg H. Van Lam van T. Pilgram O. Moulton H. Stein D.A. Hardes K. Eickmann M. Dolnik O. et al.TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells.Life Sci Alliance. 2020; 3: e202000786Crossref PubMed Google Scholar; Hoffmann et al., 2020Hoffmann M. Kleine-Weber H. Pohlmann S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells.Mol. Cell. 2020; 78: 779-784.e5Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar; Matsuyama et al., 2010Matsuyama S. Nagata N. Shirato K. Kawase M. Takeda M. Taguchi F. Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2.J. Virol. 2010; 84: 12658-12664Crossref PubMed Scopus (444) Google Scholar; Örd et al., 2020Örd M. Faustova I. Loog M. The sequence at Spike S1/S2 site enables cleavage by furin and phospho-regulation in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV.Sci. Rep. 2020; 10: 16944Crossref PubMed Scopus (43) Google Scholar; Tang et al., 2021Tang T. Jaimes J.A. Bidon M.K. Straus M.R. Daniel S. Whittaker G.R. Proteolytic Activation of SARS-CoV-2 Spike at the S1/S2 Boundary: Potential Role of Proteases beyond Furin.ACS Infect. Dis. 2021; 7: 264-272Crossref PubMed Scopus (52) Google Scholar). The S1/S2 cleavage exposes the S2′ site, and a second cleavage of the S2′ is needed to release an internal fusion peptide that mediates membrane fusion (Xia et al., 2020Xia S. Zhu Y. Liu M. Lan Q. Xu W. Wu Y. Ying T. Liu S. Shi Z. Jiang S. Lu L. Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein.Cell. Mol. Immunol. 2020; 17: 765-767Crossref PubMed Scopus (340) Google Scholar). Since 2019, several SARS-CoV-2 lineages have emerged, leading to the divergence of an extensive subset of SARS-CoV-2 variants termed “variants of concern” (VOCs). This has led to the natural selection of several mutations in the S protein with different functional consequences, some of them unknown. As SARS-CoV-2 variants are arising, more research is needed to understand what the drivers of evolutionary changes are over time and the potential impact on epidemiology, antigenicity, escape from neutralizing antibodies induced by previous infection, and vaccination and virus fitness. The first widely adaptative substitution described was the S protein amino acid change S:D614G, which became dominant in March 2020 and is present in most of the variants currently circulating worldwide. This substitution is known to enhance viral replication in the upper respiratory tract as well as in vivo transmission (Korber et al., 2020Korber B. Fischer W.M. Gnanakaran S. Yoon H. Theiler J. Abfalterer W. Hengartner N. Giorgi E.E. Bhattacharya T. Foley B. et al.Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.Cell. 2020; 182: 812-827.e19Abstract Full Text Full Text PDF PubMed Scopus (1817) Google Scholar; Plante et al., 2021Plante J.A. Liu Y. Liu J. Xia H. Johnson B.A. Lokugamage K.G. Zhang X. Muruato A.E. Zou J. Fontes-Garfias C.R. et al.Spike mutation D614G alters SARS-CoV-2 fitness.Nature. 2021; 592: 116-121Crossref PubMed Scopus (604) Google Scholar). Several other polymorphisms became dominant in late 2020. The N501Y substitution convergently evolved in emerging VOCs alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and omicron (B.1.1.529) variants and has been associated with an enhanced spike affinity for the cellular ACE2 receptor (Liu et al., 2021Liu Y. Liu J. Plante K.S. Plante J.A. Xie X. Zhang X. Ku Z. An Z. Scharton D. Schindewolf C. et al.The N501Y spike substitution enhances SARS-CoV-2 transmission.Nature. 2021; (Published online November 24, 2021)https://doi.org/10.1038/s41586-021-04245-0Crossref Scopus (36) Google Scholar; Yi et al., 2020Yi C. Sun X. Ye J. Ding L. Liu M. Yang Z. Lu X. Zhang Y. Ma L. Gu W. et al.Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies.Cell. Mol. Immunol. 2020; 17: 621-630Crossref PubMed Scopus (208) Google Scholar). This mutation is located in the receptor-binding motif of the RBD, the primary target of many neutralizing antibodies. Importantly, accumulation of mutations in the RBD can decrease neutralizing antibody responses elicited by infection or vaccination against ancestral SARS-CoV-2 variants (Aydillo et al., 2021Aydillo T. Rombauts A. Stadlbauer D. Aslam S. Abelenda-Alonso G. Escalera A. Amanat F. Jiang K. Krammer F. Carratala J. García-Sastre A. Immunological imprinting of the antibody response in COVID-19 patients.Nat. Commun. 2021; 12: 3781Crossref PubMed Scopus (34) Google Scholar). Similarly, the later SARS-CoV-2 kappa (B.1.617.1) and delta (B.1.617.2) variants have also shown a significantly reduced sensitivity to convalescent and immune sera (Edara et al., 2021Edara V.V. Lai L. Sahoo M.K. Floyd K. Sibai M. Solis D. Flowers M.W. Hussaini L. Ciric C.R. Bechnack S. et al.Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant.Prepint at bioRxiv. 2021; https://doi.org/10.1101/2021.05.09.443299Crossref Google Scholar; Mlcochova et al., 2021aMlcochova P. Kemp S. Dhar M.S. Papa G. Meng B. Mishra S. Whittaker C. Mellan T. Ferreira I. Datir R. et al.SARS-CoV-2 B.1.617.2 Delta variant emergence, replication and sensitivity to neutralising antibodies.Preprint at bioRxiv. 2021; https://doi.org/10.1101/2021.05.08.443253Crossref Scopus (0) Google Scholar; Planas et al., 2021bPlanas D. Veyer D. Baidaliuk A. Staropoli I. Guivel-Benhassine F. Rajah M.M. Planchais C. Porrot F. Robillard N. Puech J. et al.Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization.Nature. 2021; 596: 276-280Crossref PubMed Scopus (648) Google Scholar). Other mutations outside the RBD have also become prevalent. A clear example is the polymorphism found at position S:681 in the furin cleavage site, which includes P681H and P681R in the alpha/omicron and kappa/delta variants, respectively. Some preliminary reports have pointed to an enhancement in virus transmissibility associated with this polymorphism, perhaps due to an increase of spike cleavage (Peacock et al., 2021Peacock T.P. Sheppard C.M. Brown J.C. Goonawardane N. Zhou J. Whiteley M. Consortium P.V. de Silva T.I. Barclay W.S. The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin.Preprint at bioRxiv. 2021; https://doi.org/10.1101/2021.05.28.446163Crossref Scopus (0) Google Scholar). Additionally, several other mutations have been identified at the edge of the furin cleavage site. This is the case of the H655Y substitution found in the gamma (P.1) and omicron variants (B.1.1.529). This mutation was associated with changes in antigenicity by conferring escape from human monoclonal antibodies (Baum et al., 2020Baum A. Fulton B.O. Wloga E. Copin R. Pascal K.E. Russo V. Giordano S. Lanza K. Negron N. Ni M. et al.Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies.Science. 2020; 369: 1014-1018Crossref PubMed Scopus (577) Google Scholar). Moreover, it has also been found to be selected in animal models after experimental infection in vivo (Braun et al., 2021Braun K.M. Moreno G.K. Halfmann P.J. Hodcroft E.B. Baker D.A. Boehm E.C. Weiler A.M. Haj A.K. Hatta M. Chiba S. et al.Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.PLoS Pathog. 2021; 17: e1009373Crossref PubMed Google Scholar; Rathnasinghe et al., 2021Rathnasinghe R. Jangra S. Cupic A. Martinez-Romero C. Mulder L.C.F. Kehrer T. Yildiz S. Choi A. Mena I. De Vrieze J. et al.The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera.Preprint at medRxiv. 2021; https://doi.org/10.1101/2021.01.19.21249592Crossref Scopus (0) Google Scholar), indicating a potential role in host replication, transmissibility, and pathogenicity. Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. Using the mink model of COVID-19, we found that the S:H655Y substitution was acquired in vivo after infection with the WA1 isolate (USA-WA1/2020). To investigate the advantage conferred by S:H655Y, we analyzed the kinetics, spike processing by cellular proteases, and syncytium formation ability of a panel of SARS-CoV-2 variants harboring 655Y, including human isolates derived from patients seeking care at the Mount Sinai Health System in New York (NY) City, which was one of the major early epicenters of the COVID-19 pandemic. Our results demonstrate that the 655Y polymorphism enhances spike cleavage and viral growth. Furthermore, the S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to overcome S:655H in the human airway epithelial system. Finally, and in the context of the current epidemiological situation, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. We demonstrate that novel circulating VOCs that have become more prevalent have independently acquired mutations associated with a gain in spike cleavage and syncytia formation. Taken together, our study shows a link between an increased spike processing and increased virus transmission due to the spike mutations present in SARS-CoV-2 variants that have become epidemiologically more prevalent in humans. Minks have been suggested to play a role in the initial local spread and evolution of SARS-CoV-2 variants in different countries in Europe (Hammer et al., 2021Hammer A.S. Quaade M.L. Rasmussen T.B. Fonager J. Rasmussen M. Mundbjerg K. Lohse L. Strandbygaard B. Jørgensen C.S. Alfaro-Núñez A. et al.SARS-CoV-2 Transmission between Mink (Neovison vison) and Humans, Denmark.Emerg. Infect. Dis. 2021; 27: 547-551Crossref PubMed Scopus (91) Google Scholar; Oude Munnink et al., 2021Oude Munnink B.B. Sikkema R.S. Nieuwenhuijse D.F. Molenaar R.J. Munger E. Molenkamp R. van der Spek A. Tolsma P. Rietveld A. Brouwer M. et al.Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans.Science. 2021; 371: 172-177Crossref PubMed Scopus (376) Google Scholar). While minks are susceptible to SARS-CoV-2, they are also capable for zoonotic transmission of SARS-CoV-2 because of the similarity of the ACE2 receptor between minks and humans. We used the mink model to investigate the replication and pathogenicity of the WA1 (USA-WA1/2020) isolate of SARS-CoV-2 as a representative of the first original human viruses that initiated the SARS-CoV-2 pandemic. This variant corresponds to one of the first isolates of the United States of America (USA) and does not contain any changes on the S protein when compared to the initial isolates from Wuhan, such as the Wuhan-1 virus. For this purpose, 6 minks were intranasally infected with 106 pfu of WA1 isolate, resulting in productive viral replication in the upper respiratory tract with infectious virus recovered from nasal washes at days 1, 3, and 5 post-infection (p.i.) (Figures S1A and S1B). At day 4 post-inoculation, infectious virus was detected by plaque assays from left cranial lung and nasal turbinates, but not from any of the other tissues analyzed (Figure S1C). We then selected small and large viral plaques in the Vero E6 cell-based plaque assays from infected mink lung specimens and performed next generation sequencing of the genome from the plaque-isolated viruses. As compared to the Wuhan-1 and WA1 reference sequences, all mink-derived viral isolates encoded the H655Y amino acid substitution within S (Figure S1D). Additionally, the 3 viral isolates with the small plaque phenotype encoded the T259K amino acid substitution while the 3 viral isolates with the large plaque phenotype encoded the R682W amino acid substitution. It is known that S:682W/Q substitution in the furin cleavage site region may emerge after subsequent passages in Vero E6 cells (Lamers et al., 2021Lamers M.M. Mykytyn A.Z. Breugem T.I. Wang Y. Wu D.C. Riesebosch S. van den Doel P.B. Schipper D. Bestebroer T. Wu N.C. Haagmans B.L. Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation.eLife. 2021; 10: e66815Crossref PubMed Google Scholar; Liu et al., 2020Liu Z. Zheng H. Lin H. Li M. Yuan R. Peng J. Xiong Q. Sun J. Li B. Wu J. et al.Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.J. Virol. 2020; 94 (e00790-20)Crossref Scopus (69) Google Scholar). Next generation sequencing analysis of the viral quasispecies population in mink lung specimens and WA1 stock demonstrated the presence of around 30% of the S:682W/Q substitution, indicating that this mutation was selected during the course of the Vero E6 infections and stock generation and not during the infection in minks. On the other hand, S:655Y was present in less than 10% of the WA1 original stock, but it was dominant in all the mink isolates in 99% of the viral RNA obtained from the lungs, suggesting that this mutation was selected and may confer an advantage in the mink host. To understand the magnitude and the spread of the 655Y polymorphism over time, we investigated the frequency of S:655Y in sequences sampled worldwide since the initial outbreak to the end of the first wave of SARS-CoV-2 (Figure 1A). For this, 7,059 sequences sampled from GISAID up to September 2020 were used. Human variants harboring the 655Y mutation were spread throughout the phylogenetic tree and distributed in all clades with no differences according to temporal distribution, suggesting that the 655Y mutation arose independently multiple times. Remarkably, the S:H655Y polymorphism was also found among the initial variants introduced in NY City in March 2020. To determine the replication phenotype, we decided to investigate this NY 655Y variant (NY7) together with some of its contemporaneous SARS-CoV-2 isolates circulating in NY during the early pandemic outbreak (Gonzalez-Reiche et al., 2020Gonzalez-Reiche A.S. Hernandez M.M. Sullivan M.J. Ciferri B. Alshammary H. Obla A. Fabre S. Kleiner G. Polanco J. Khan Z. et al.Introductions and early spread of SARS-CoV-2 in the New York City area.Science. 2020; 369: 297-301Crossref PubMed Scopus (157) Google Scholar). To this end, we isolated 12 viruses based on their genotypes (Gonzalez-Reiche et al., 2020Gonzalez-Reiche A.S. Hernandez M.M. Sullivan M.J. Ciferri B. Alshammary H. Obla A. Fabre S. Kleiner G. Polanco J. Khan Z. et al.Introductions and early spread of SARS-CoV-2 in the New York City area.Science. 2020; 369: 297-301Crossref PubMed Scopus (157) Google Scholar), including NY7, which carries the S:655Y mutation for culture directly from nasopharyngeal specimens obtained from COVID-19-infected patients. Of note, the dominant 614G spike polymorphism was present in seven (NY1, NY2, NY3, NY4, NY9, NY10, and NY12; 58%) of the selected human SARS-CoV-2 (hCoV) NY isolates, which is consistent with its early emergence and rapid spread worldwide (Korber et al., 2020Korber B. Fischer W.M. Gnanakaran S. Yoon H. Theiler J. Abfalterer W. Hengartner N. Giorgi E.E. Bhattacharya T. Foley B. et al.Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.Cell. 2020; 182: 812-827.e19Abstract Full Text Full Text PDF PubMed Scopus (1817) Google Scholar; Plante et al., 2021Plante J.A. Liu Y. Liu J. Xia H. Johnson B.A. Lokugamage K.G. Zhang X. Muruato A.E. Zou J. Fontes-Garfias C.R. et al.Spike mutation D614G alters SARS-CoV-2 fitness.Nature. 2021; 592: 116-121Crossref PubMed Scopus (604) Google Scholar). Confirmation sequencing of the isolates showed that 682W/Q substitutions appeared in 4 (33%) viruses after initial isolation and culturing in Vero E6 cells. However, this mutation was absent in the original nasal swabs collected from the corresponding COVID-19-infected patients. This is consistent with in vitro adaptative mutations previously described (Lamers et al., 2021Lamers M.M. Mykytyn A.Z. Breugem T.I. Wang Y. Wu D.C. Riesebosch S. van den Doel P.B. Schipper D. Bestebroer T. Wu N.C. Haagmans B.L. Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation.eLife. 2021; 10: e66815Crossref PubMed Google Scholar). Moreover, a 5-amino-acid sequence (Δ675–679) flanking the furin cleavage site was deleted in five (42%) of the isolates as compared to the sequence from the original specimen. This deletion has been previously reported to be a common in vitro mutation selected in Vero cells (Liu et al., 2020Liu Z. Zheng H. Lin H. Li M. Yuan R. Peng J. Xiong Q. Sun J. Li B. Wu J. et al.Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.J. Virol. 2020; 94 (e00790-20)Crossref Scopus (69) Google Scholar). Amino acids substitutions of the S protein of these initial human isolates compared to the Wuhan-1 reference are shown in Figure S2A and Table S2. We next studied the replication kinetics of the NY SARS-CoV-2 isolates by comparing their multicycle growth curves at an MOI of 0.01 in Vero E6 and human Caco-2 cells. As expected, NY2, NY4, and NY9 containing the 682Q/W showed an advantage in growth at 48 h p.i. in Vero E6 cells, while no differences could be found in Caco-2 cells (Figures S2B–S2E). Remarkably, NY7 (S:655Y) showed higher growth at 48 h p.i in Caco-2 cells (Figures S2C and S2E) when compared to the rest of these early SARS-CoV-2 isolates. These results support our conclusion that the 655Y polymorphism conferred a viral advantage. To investigate the spike cleavage efficiency of the 655Y versus other human isolates, we performed infections in Vero E6, and supernatants were analyzed by western blot for the S2 domain of the S protein. Importantly, 2 bands were clearly visible for the NY7 (S:655Y) (Figure 1B), corresponding to both the cleaved (95 kDa) and uncleaved (180 kDa) form of the S protein. In contrast, subtle bands for S2 were detected in NY6, NY10, and NY12, and only the uncleaved S form was detected in the other early human isolates. Consistently, quantification of full-length and cleaved S protein showed that around 80% of the NY7 (S:655Y) S was in its S2 cleaved form (Figure 1C), indicating that the 655Y polymorphism may facilitate S protein processing. To confirm these results in a primary human cell model, we assessed the S protein processing of the twelve early NY human isolates in pneumocyte-like cells. This system has been previously described as a robust model to study SARS-CoV-2 infection (Riva et al., 2020Riva L. Yuan S. Yin X. Martin-Sancho L. Matsunaga N. Pache L. Burgstaller-Muehlbacher S. De Jesus P.D. Teriete P. Hull M.V. et al.Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.Nature. 2020; 586: 113-119Crossref PubMed Scopus (343) Google Scholar; White et al., 2021White K.M. Rosales R. Yildiz S. Kehrer T. Miorin L. Moreno E. Jangra S. Uccellini M.B. Rathnasinghe R. Coughlan L. et al.Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.Science. 2021; 371: 926-931Crossref PubMed Scopus (96) Google Scholar). Human pneumocyte-like cells were differentiated from human induced pluripotent stem cell and infected with the NY SARS-CoV-2 isolates. Cell extracts were collected at 48 h p.i and analyzed by western blot (Figure 1D). As expected, NY7 (S:655Y) showed an increase cleavage efficiency with 70% of cleaved S protein (Figure 1E). Additionally, we observed S2 cleaved protein in NY12 consistent with the S2 subtle bands detected in Vero E6 cells. This isolate only contains the S:D614G mutation. Although other NY variants used in this study also harbor the D614G, no enhancement in spike cleavage was observed. This can be explained by the acquisition of additional S mutations during in vitro culture (Δ675–679, 682Q/W) in the isolates containing the D614G, which could be masking the effect of D614G in spike processing. To confirm whether the 655Y mutation was solely responsible for the increased S cleavage, we analyzed the replication and cleavage efficiency of a panel of SARS-CoV-2 viruses, all bearing the 655Y substitution but containing additional substitutions across the genome. We included two of the isolated mink variants (MiA1 and MiA2), NY7 and another human isolate derived from a COVID-19 patient infected in February 2021 (NY13, S:614G, 655Y), and a WA1-655Y variant isolated after wild-type WA1 infection in Vero E6 cells and subsequent plaque purification. Additionally, the WA1 reference and NY6 were used as controls since they lack the 655Y substitution. It should be noted that NY6 has a 5-amino-acid deletion before the furin cleavage site (Figures 2A and S2A). We assessed differences in replication and S processing of this panel of viruses by comparing growth in both Vero E6 and Vero-TMPRSS2 cells. As shown in Figure 2B, WA1-655Y infection yielded higher titers in both Vero E6 and Vero-TMPRSS2 cells as compared to infection by WA1 (statistically significant in Vero E6 at 24 h p.i.; Table S4). These isolates only differ in the position 655Y while the rest of the genome is isogenic, supporting that 655Y spike polymorphism enhances viral replication and growth. Next, viral supernatants were used to analyze the plaque phenotype in Vero E6 and Vero-TMPRSS2 and to compare S protein expression levels after infection. In general, all isolates showed higher plaque size in the presence of TMPRSS2, consistent with enhancement of cell entry (Figure S3A). However, differences were found by western blot, and only the isolates bearing 655Y showed enhanced spike cleavage in both Vero E6 and Vero-TMPRSS2 (Figure 2C). More than 90% of the total S protein from these 655Y variants corresponded to the S2 cleaved form of the spike. In contrast, NY6 and WA1 controls showed poor cleavage efficiency (Figures 2D and 2E). Finally, we performed infections with a representative panel of viruses containing the 655Y (NY7, NY13, WA1-655Y, NY6, and WA1) in human pneumocyte-like cells to assess viral growth and S protein processing. Consistent with our previous findings, WA1-655Y demonstrated higher replication efficiency in our human airway epithelial system compared to WA1 wild type (Figure 2F; Table S4). Moreover, all isolates encoding the 655Y spike mutation exhibited enhanced spike cleavage as shown by western blot (Figures 2G and 2H). This demonstrates that the S:655Y polymorphism plays a crucial role in SARS-CoV-2 S protein processing and cell entry in human pneumocyte-like cells. Syncytia formation has been described as one of the hallmarks of SARS-CoV-2 infection and pathogenesis in the lungs. It is mediated by the interaction
95 citations
TL;DR: In this paper , the authors demonstrate that recently emerged BQ.1.1 and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants.
Abstract: Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots1. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.
15 citations
TL;DR: In this article, the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants was investigated, and it was shown that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility.
Abstract: For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2 by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.
9 citations
TL;DR: This work generated and characterized a panel of anti-RBD monoclonal antibodies (MAbs) isolated from eukaryotic recombinant RBD-immunized mice by hybridoma technology, and found that the neutralizing MAbs recognize mainly conformational epitopes, largely supported by the Western blotting results.
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the outbreak led to the coronavirus disease 2019 (COVID-19) pandemic. Receptor binding domain (RBD) of spike (S) protein of SARS-CoV-2 is considered as a major target for immunotherapy and vaccine design. Here, we generated and characterized a panel of anti-RBD monoclonal antibodies (MAbs) isolated from eukaryotic recombinant RBD-immunized mice by hybridoma technology. Epitope mapping was performed using a panel of 20-mer overlapping peptides spanning the entire sequence of the RBD protein from wild-type (WT) Wuhan strain by enzyme-linked immunosorbent assay (ELISA). Several hybridomas showed reactivity toward restricted RBD peptide pools by Pepscan analysis, with more focus on peptides encompassing aa 76–110 and 136–155. However, our MAbs with potent neutralizing activity which block SARS-CoV-2 spike pseudovirus as well as the WT virus entry into angiotensin-converting enzyme-2 (ACE2) expressing HEK293T cells showed no reactivity against these peptides. These findings, largely supported by the Western blotting results suggest that the neutralizing MAbs recognize mainly conformational epitopes. Moreover, our neutralizing MAbs recognized the variants of concern (VOC) currently in circulation, including alpha, beta, gamma, and delta by ELISA, and neutralized alpha and omicron variants at different levels by conventional virus neutralization test (CVNT). While the neutralization of MAbs to the alpha variant showed no substantial difference as compared with the WT virus, their neutralizing activity was lower on omicron variant, suggesting the refractory effect of mutations in emerging variants against this group of neutralizing MAbs. Also, the binding reactivity of our MAbs to delta variant showed a modest decline by ELISA, implying that our MAbs are insensitive to the substitutions in the RBD of delta variant. Our data provide important information for understanding the immunogenicity of RBD, and the potential application of the novel neutralizing MAbs for passive immunotherapy of SARS-CoV-2 infection.
4 citations
TL;DR: In this paper, nine receptor-binding domain (RBD) proteins of the SARS-CoV-2 variants (B.1.7, B.351 and P.1 lineages) were constructed and fused with the Fc fragment of human IgG, which enabled them to bind strongly to the polyclonal antibodies specific for wild-type RBD and to recombinant human ACE2 protein.
3 citations
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TL;DR: The epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of patients with laboratory-confirmed 2019-nCoV infection in Wuhan, China, were reported.
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TL;DR: Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily, which is the seventh member of the family of coronaviruses that infect humans.
Abstract: In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).
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TL;DR: It is demonstrated that SARS-CoV-2 uses the SARS -CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming, and it is shown that the sera from convalescent SARS patients cross-neutralized Sars-2-S-driven entry.
15,362 citations
TL;DR: The authors show that this protein binds at least 10 times more tightly than the corresponding spike protein of severe acute respiratory syndrome (SARS)–CoV to their common host cell receptor, and test several published SARS-CoV RBD-specific monoclonal antibodies found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs.
Abstract: The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.
7,324 citations
TL;DR: CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations in the third major release of RELION.
Abstract: Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 A compared to previous RELION versions.
3,520 citations