SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses

AbstractThe majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.

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A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies

Journal of General Virology ◽

10.1099/vir.0.80844-0 ◽

2005 ◽

Vol 86 (5) ◽

pp. 1435-1440 ◽

Cited By ~ 65

Author(s):

Milosz Faber ◽

Elaine W. Lamirande ◽

Anjeanette Roberts ◽

Amy B. Rice ◽

Hilary Koprowski ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Protein S ◽

S Protein ◽

Cellular Immune Responses ◽

Glycoprotein G ◽

Cellular Immune ◽

Animal Reservoirs ◽

S Genes

Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.

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Structures of Two Human Astrovirus Capsid/Neutralizing Antibody Complexes Reveal Distinct Epitopes and Inhibition of Virus Attachment to Cells

Journal of Virology ◽

10.1128/jvi.01415-21 ◽

2021 ◽

Author(s):

Lena Ricemeyer ◽

Nayeli Aguilar-Hernández ◽

Tomás López ◽

Rafaela Espinosa ◽

Sarah Lanning ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Severe Disease ◽

Neutralizing Antibody ◽

Host Cells ◽

Viral Gastroenteritis ◽

Receptor Binding Site ◽

Virus Attachment ◽

Human Astroviruses ◽

Human Astrovirus

Human astrovirus is an important cause of viral gastroenteritis worldwide. Young children, the elderly, and the immunocompromised are especially at risk for contracting severe disease. However, no vaccines exist to combat human astrovirus infection. Evidence points to the importance of antibodies in enabling protection of healthy adults from reinfection. To develop an effective subunit vaccine that broadly protects against diverse astrovirus serotypes, we must understand how neutralizing antibodies target the capsid surface at the molecular level. Here, we report the structures of the human astrovirus capsid spike domain bound to two neutralizing monoclonal antibodies. These antibodies bind two distinct conformational epitopes on the spike surface. We add to existing evidence that the human astrovirus capsid spike contains a receptor-binding domain and demonstrate that both antibodies neutralize human astrovirus by blocking virus attachment to host cells. We identify patches of conserved amino acids that overlap or border the antibody epitopes and may constitute a receptor-binding site. Our findings provide a basis to develop therapies that prevent and treat human astrovirus gastroenteritis. Importance Human astroviruses infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies block virus infection. Here, we determined crystal structures of the astrovirus capsid protein in complex with two virus-neutralizing antibodies. We show that the antibodies bind two distinct sites on the capsid spike domain; however, both antibodies block virus attachment to human cells. Importantly, our findings support the use of the human astrovirus capsid spike as an antigen in a subunit-based vaccine to prevent astrovirus disease.

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Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization

10.1101/2021.08.25.457627 ◽

2021 ◽

Author(s):

Yunjeong Kim ◽

Natasha N Gaudreault ◽

David A Meekins ◽

Krishani D Perera ◽

Dashzeveg Bold ◽

...

Keyword(s):

Viral Entry ◽

Neutralizing Antibodies ◽

Animal Species ◽

Virus Entry ◽

Neutralizing Antibody ◽

Protein S ◽

Pseudotyped Virus ◽

S Protein ◽

Viral Neutralization ◽

Wide Range

SARS-CoV-2 is a zoonotic agent capable of infecting humans and a wide range of animal species. Over the duration of the pandemic, mutations in the SARS-CoV-2 Spike protein (S) have arisen in circulating viral populations, culminating in the spread of several variants of concern (VOC) with varying degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we employed lentivirus-based pseudotyped viruses that express specific SARS-CoV-2 S protein substitutions and cell lines that stably express ACE2 from nine different animal species to gain insights into the effects of VOC mutations on viral entry and antibody neutralization capability. All animal ACE2 receptors tested, except mink, support viral cell entry for pseudoviruses expressing the parental (prototype Wuhan-1) S at levels comparable to human ACE2. Most single S substitutions (e.g., 452R, 478K, 501Y) did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency in viral entry. Conversely, combinatorial VOC substitutions in the S protein were associated with significantly increased entry capacity of pseudotyped viruses compared to that of the parental Wuhan-1 pseudotyped virus. Similarly, infection studies using live ancestral (USA-WA1/2020), Alpha, and Beta SARS-CoV-2 viruses in hamsters revealed a higher replication potential for the Beta variant compared to the ancestral prototype virus. Moreover, neutralizing titers in sera from various animal species, including humans, were significantly reduced by single substitutions of 484K or 452R, double substitutions of 501Y-484K, 452R-484K and 452R-478K and the triple substitution of 501Y-484K-417N, suggesting that 484K and 452R are particularly important for evading neutralizing antibodies in human, cat, and rabbit sera. Cumulatively, this study reveals important insights into the host range of SARS-CoV-2 and the effect of recently emergent S protein substitutions on viral entry, virus replication and antibody-mediated viral neutralization.

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Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic

Antibody Therapeutics ◽

10.1093/abt/tbaa025 ◽

2020 ◽

Vol 3 (4) ◽

pp. 246-256

Author(s):

Yaping Sun ◽

Mitchell Ho

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Host Cells ◽

Spike Protein ◽

Inhaled Drugs ◽

Viral Attachment ◽

Global Pandemic ◽

Attachment Sites ◽

Antibody Discovery

Abstract SARS-CoV-2 antibody therapeutics are being evaluated in clinical and preclinical stages. As of 11 October 2020, 13 human monoclonal antibodies targeting the SARS-CoV-2 spike protein have entered clinical trials with three (REGN-COV2, LY3819253/LY-CoV555, and VIR-7831/VIR-7832) in phase 3. On 9 November 2020, the US Food and Drug Administration issued an emergency use authorization for bamlanivimab (LY3819253/LY-CoV555) for the treatment of mild-to-moderate COVID-19. This review outlines the development of neutralizing antibodies against SARS-CoV-2, with a focus on discussing various antibody discovery strategies (animal immunization, phage display and B cell cloning), describing binding epitopes and comparing neutralizing activities. Broad-neutralizing antibodies targeting the spike proteins of SARS-CoV-2 and SARS-CoV might be helpful for treating COVID-19 and future infections. VIR-7831/7832 based on S309 is the only antibody in late clinical development, which can neutralize both SARS-CoV-2 and SARS-CoV although it does not directly block virus receptor binding. Thus far, the only cross-neutralizing antibody that is also a receptor binding blocker is nanobody VHH-72. The feasibility of developing nanobodies as inhaled drugs for treating COVID-19 and other respiratory diseases is an attractive idea that is worth exploring and testing. A cocktail strategy such as REGN-COV2, or engineered multivalent and multispecific molecules, combining two or more antibodies might improve the efficacy and protect against resistance due to virus escape mutants. Besides the receptor-binding domain, other viral antigens such as the S2 subunit of the spike protein and the viral attachment sites such as heparan sulfate proteoglycans that are on the host cells are worth investigating.

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SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

Nature Communications ◽

10.1038/s41467-020-19808-4 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 5

Author(s):

Lizhou Zhang ◽

Cody B. Jackson ◽

Huihui Mou ◽

Amrita Ojha ◽

Haiyong Peng ◽

...

Keyword(s):

Protein Binding ◽

Protein S ◽

Spike Protein ◽

Neutralization Sensitivity ◽

Spike Density ◽

S Protein ◽

Virus Like Particles ◽

Protein Incorporation ◽

S Proteins

AbstractSARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (SG614) with the original (SD614). We report here pseudoviruses carrying SG614 enter ACE2-expressing cells more efficiently than those with SD614. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.

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Cryo-EM Structures of the N501Y SARS-CoV-2 Spike Protein in Complex with ACE2 and Two Potent Neutralizing Antibodies

10.1101/2021.01.11.426269 ◽

2021 ◽

Cited By ~ 1

Author(s):

Xing Zhu ◽

Dhiraj Mannar ◽

Shanti S. Srivastava ◽

Alison M. Berezuk ◽

Jean-Philippe Demers ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Structural Changes ◽

Neutralizing Antibody ◽

Antibody Fragments ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Binding Interface ◽

Short Summary

AbstractThe recently reported “UK variant” of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments.Short summaryThe N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.

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Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques

10.1101/2021.07.05.451222 ◽

2021 ◽

Author(s):

Wan-ting He ◽

Meng Yuan ◽

Sean Callaghan ◽

Rami Musharrafieh ◽

Ge Song ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Evaluation Studies ◽

Neutralizing Antibody ◽

Primate Species ◽

Vaccine Response ◽

Cell Repertoire ◽

Receptor Binding Site ◽

Human Antibodies

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross-neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use.

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A thermostable, closed, SARS-CoV-2 spike protein trimer

10.1101/2020.06.15.152835 ◽

2020 ◽

Cited By ~ 3

Author(s):

Xiaoli Xiong ◽

Kun Qu ◽

Katarzyna A. Ciazynska ◽

Myra Hosmillo ◽

Andrew P. Carter ◽

...

Keyword(s):

Immune System ◽

Receptor Binding ◽

Conformational Flexibility ◽

Cell Entry ◽

Spike Protein ◽

Receptor Binding Site ◽

S Protein ◽

Vaccine Candidates ◽

Diagnostic Assays ◽

Potential Applications

AbstractThe spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

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Human coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-binding site in spike protein domain A

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809667116 ◽

2019 ◽

Vol 116 (7) ◽

pp. 2681-2690 ◽

Cited By ~ 117

Author(s):

Ruben J. G. Hulswit ◽

Yifei Lang ◽

Mark J. G. Bakkers ◽

Wentao Li ◽

Zeshi Li ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Receptor Binding ◽

Protein S ◽

Protein Domain ◽

Spike Protein ◽

Ligand Docking ◽

Respiratory Pathogens ◽

Receptor Binding Site ◽

Human Coronaviruses

Human betacoronaviruses OC43 and HKU1 are endemic respiratory pathogens and, while related, originated from independent zoonotic introductions. OC43 is in fact a host-range variant of the species Betacoronavirus-1, and more closely related to bovine coronavirus (BCoV)—its presumptive ancestor—and porcine hemagglutinating encephalomyelitis virus (PHEV). The β1-coronaviruses (β1CoVs) and HKU1 employ glycan-based receptors carrying 9-O-acetylated sialic acid (9-O-Ac-Sia). Receptor binding is mediated by spike protein S, the main determinant of coronavirus host specificity. For BCoV, a crystal structure for the receptor-binding domain S1A is available and for HKU1 a cryoelectron microscopy structure of the complete S ectodomain. However, the location of the receptor-binding site (RBS), arguably the single-most important piece of information, is unknown. Here we solved the 3.0-Å crystal structure of PHEV S1A. We then took a comparative structural analysis approach to map the β1CoV S RBS, using the general design of 9-O-Ac-Sia-binding sites as blueprint, backed-up by automated ligand docking, structure-guided mutagenesis of OC43, BCoV, and PHEV S1A, and infectivity assays with BCoV-S–pseudotyped vesicular stomatitis viruses. The RBS is not exclusive to OC43 and related animal viruses, but is apparently conserved and functional also in HKU1 S1A. The binding affinity of the HKU1 S RBS toward short sialoglycans is significantly lower than that of OC43, which we attribute to differences in local architecture and accessibility, and which may be indicative for differences between the two viruses in receptor fine-specificity. Our findings challenge reports that would map the OC43 RBS elsewhere in S1A and that of HKU1 in domain S1B.

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Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants

Science ◽

10.1126/science.abh1139 ◽

2021 ◽

pp. eabh1139

Author(s):

Meng Yuan ◽

Deli Huang ◽

Chang-Chun D. Lee ◽

Nicholas C. Wu ◽

Abigail M. Jackson ◽

...

Keyword(s):

South Africa ◽

Binding Site ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Antigenic Drift ◽

Spike Protein ◽

Binding Modes ◽

Next Generation ◽

Wild Type ◽

Receptor Binding Site

Neutralizing antibodies (nAbs) elicited against the receptor-binding site (RBS) of the spike protein of wild-type SARS-CoV-2 are generally less effective against recent variants of concern. RBS residues E484, K417 and N501 are mutated in variants first described in South Africa (B.1.351) and Brazil (P.1). We analyzed their effects on ACE2 binding and K417N and E484K mutations on nAbs isolated from COVID-19 patients. Binding and neutralization of the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2), which can both bind the RBS in alternate binding modes, are abrogated by K417N, E484K, or both. These effects can be structurally explained by their extensive interactions with RBS nAbs. However, nAbs to the more conserved, cross-neutralizing CR3022 and S309 sites were largely unaffected. The results have implications for next-generation vaccines and antibody therapies.

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