scholarly journals Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses

npj Vaccines ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Rinke Bos ◽  
Lucy Rutten ◽  
Joan E. M. van der Lubbe ◽  
Mark J. G. Bakkers ◽  
Gijs Hardenberg ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Neutralizing Antibodies ◽  
Etiologic Agent ◽  
Wild Type ◽  
Furin Cleavage ◽  
S Protein ◽  
Design Elements ◽  
Furin Cleavage Site ◽  
Prophylactic Measures

Abstract Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g., prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S2) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild-type signal peptide was best suited for the correct cleavage needed for a natively folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild-type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

scholarly journals Ad26-vector based COVID-19 vaccine encoding a prefusion stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses

2020 ◽  
Author(s):  
Rinke Bos ◽  
Lucy Rutten ◽  
Joan E.M. van der Lubbe ◽  
Mark J.G. Bakkers ◽  
Gijs Hardenberg ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Neutralizing Antibodies ◽  
Etiologic Agent ◽  
Wild Type ◽  
Furin Cleavage ◽  
S Protein ◽  
Design Elements ◽  
Furin Cleavage Site ◽  
Prophylactic Measures

AbstractDevelopment of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g. prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S1) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild type signal peptide was best suited for the correct cleavage needed for a natively-folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-γ. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

scholarly journals Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage

2021 ◽  
Author(s):  
Nikita Shiliaev ◽  
Tetyana Lukash ◽  
Oksana Palchevska ◽  
David K Crossman ◽  
Todd J. Green ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Cleavage Site ◽  
Heparin Binding ◽  
Natural Isolate ◽  
Furin Cleavage ◽  
S Protein ◽  
Viral Spread ◽  
Furin Cleavage Site ◽  
Viral Particles

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo.

scholarly journals SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients

2020 ◽  
Author(s):  
William B. Klimstra ◽  
Natasha L. Tilston-Lunel ◽  
Sham Nambulli ◽  
James Boslett ◽  
Cynthia M. McMillen ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Serum Samples ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Similar Disease ◽  
Antibody Titers ◽  
Low Passage

AbstractSARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

scholarly journals Broad and differential animal ACE2 receptor usage by SARS-CoV-2

2020 ◽  
Author(s):  
Xuesen Zhao ◽  
Danying Chen ◽  
Robert Szabla ◽  
Mei Zheng ◽  
Guoli Li ◽  
...  
Keyword(s):  
Animal Models ◽  
Host Range ◽  
Cleavage Site ◽  
Receptor Activity ◽  
Host Cells ◽  
Interspecies Transmission ◽  
Wild Type ◽  
Furin Cleavage ◽  
S Protein ◽  
Furin Cleavage Site

ABSTRACTThe COVID-19 pandemic has caused an unprecedented global public health and economy crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, comparing to the SARS-CoV-2-like CoVs identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human ACE2 as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2 from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activity, respectively. Among the remaining species, ACE2 from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.ImportanceSARS-CoV-2 uses human ACE2 as primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologues and found that wild type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models and molecular basis of receptor binding for SARS-CoV-2.

scholarly journals Broad and Differential Animal Angiotensin-Converting Enzyme 2 Receptor Usage by SARS-CoV-2

2020 ◽  
Vol 94 (18) ◽  
Author(s):  
Xuesen Zhao ◽  
Danying Chen ◽  
Robert Szabla ◽  
Mei Zheng ◽  
Guoli Li ◽  
...  
Keyword(s):  
Animal Models ◽  
Angiotensin Converting Enzyme ◽  
Cleavage Site ◽  
Interspecies Transmission ◽  
Converting Enzyme ◽  
Wild Type ◽  
Furin Cleavage ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Furin Cleavage Site

ABSTRACT The COVID-19 pandemic has caused an unprecedented global public health and economic crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, unlike the SARS-CoV-2-like coronaviruses (CoVs) identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2s from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site-deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activities, respectively. Among the remaining species, ACE2s from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models. IMPORTANCE SARS-CoV-2 uses human ACE2 as a primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologs and found that wild-type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models, and molecular basis of receptor binding for SARS-CoV-2.

scholarly journals Trypsin Enhances SARS-CoV-2 Infection by Facilitating Viral Entry

2021 ◽  
Author(s):  
Yeeun Kim ◽  
Guehwan Jang ◽  
Duri Lee ◽  
Nara Kim ◽  
Jeong Won Seon ◽  
...  
Keyword(s):  
Viral Entry ◽  
Cultured Cells ◽  
Tissue Injury ◽  
Furin Cleavage ◽  
S Protein ◽  
Endosomal Membrane ◽  
Furin Cleavage Site ◽  
Target Organs ◽  
Syncytia Formation

Abstract Coronavirus infects the cell by cytoplasmic or endosomal membrane fusion driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and S2′ site by cellular proteases. Exogenous trypsin as a medium additive facilitates isolation and propagation of several coronaviruses in vitro. Here, we showed that trypsin mediated the enhancement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cultured cells and that SARS-CoV-2 entered the cells via either a non-endosomal or an endosomal fusion pathway depending on the presence of trypsin. Interestingly, trypsin enabled viral entry at the cell surface and led to more efficient infection efficiency than trypsin-independent endosomal entry, suggesting that trypsin production in the target organs may trigger high replication of SARS-CoV-2 and cause severe tissue injury. Extensive syncytia formation and enhanced growth kinetics were observed only in the presence of exogenous trypsin for the cell-adapted SARS-CoV-2 strains. During 50 serial passages without trypsin addition, a specific R685S mutation occurred in the S1/S2 FCS (681PRRAR685) that was completely conserved but with several mutations in the S2 fusion subunit in the presence of trypsin. These findings demonstrate that S1/S2 FCS is essential for S protein proteolytic priming and fusion activity for SARS-CoV-2 entry but not for SARS-CoV-2 replication. Our data can contribute to the improvement of SARS-CoV-2 production to develop vaccines or antivirals and motivate further investigations into the explicit functions of cell adaptation-related genetic drift in SARS-CoV-2 pathogenesis.

scholarly journals The S2 Subunit of QX-type Infectious Bronchitis Coronavirus Spike Protein Is an Essential Determinant of Neurotropism

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 972 ◽  
Author(s):  
Jinlong Cheng ◽  
Ye Zhao ◽  
Gang Xu ◽  
Keran Zhang ◽  
Wenfeng Jia ◽  
...  
Keyword(s):  
Neutralization Test ◽  
Protein S ◽  
Spike Protein ◽  
Wild Type ◽  
Effective Protection ◽  
Furin Cleavage ◽  
Infectious Bronchitis ◽  
Furin Cleavage Site

Some coronaviruses (CoVs) have an extra furin cleavage site (RRKR/S, furin-S2′ site) upstream of the fusion peptide in the spike protein, which plays roles in virion adsorption and fusion. Mutation of the S2′ site of QX genotype (QX-type) infectious bronchitis virus (IBV) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2′ site (furin-S2′ site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood–brain barrier. The results of a neutralization test and immunoprotection experiment show that an original serum and vaccine can still provide effective protection in vivo and in vitro. This is the first demonstration of IBV-induced neural symptoms in chickens with encephalitis and the furin-S2′ site as a determinant of neurotropism.

scholarly journals SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients

2020 ◽  
Vol 101 (11) ◽  
pp. 1156-1169 ◽  
Author(s):  
William B. Klimstra ◽  
Natasha L. Tilston-Lunel ◽  
Sham Nambulli ◽  
James Boslett ◽  
Cynthia M. McMillen ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Serum Samples ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Antibody Titres ◽  
Low Passage

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

scholarly journals The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

2021 ◽  
Vol 22 (12) ◽  
pp. 6490
Author(s):  
Olga A. Postnikova ◽  
Sheetal Uppal ◽  
Weiliang Huang ◽  
Maureen A. Kane ◽  
Rafael Villasmil ◽  
...  
Keyword(s):  
Amino Acid ◽  
Insertion Sequence ◽  
Experimental Studies ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
New Host ◽  
S Protein ◽  
Furin Cleavage Site ◽  
Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

scholarly journals Murine monoclonal antibodies against RBD 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

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but 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 whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing 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 variants in vitro.

Sign in / Sign up

Export Citation Format

Share Document