scholarly journals An engineered receptor-binding domain improves the immunogenicity of multivalent SARS-CoV-2 vaccines

2020 ◽  
Author(s):  
Brian D. Quinlan ◽  
Wenhui He ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Yan Guo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Viral Entry ◽  
Binding Domain ◽  
Vaccine Antigen ◽  
Receptor Binding Domain ◽  
Protein Vaccine ◽  
Wild Type ◽  
S Protein ◽  
H Pylori

ABSTRACTThe SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD expresses inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) expresses markedly more efficiently, and generates a more potent neutralizing responses as a DNA vaccine antigen, than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers such as an H. pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.

scholarly journals An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Yan Guo ◽  
Wenhui He ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Jing Chang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Binding Domain ◽  
Full Length ◽  
Vaccine Antigen ◽  
Receptor Binding Domain ◽  
Protein Vaccine ◽  
Wild Type ◽  
S Protein ◽  
Glycosylation Sites

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines. IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

scholarly journals LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro

2020 ◽  
Author(s):  
Annika Roth ◽  
Steffen Lütke ◽  
Denise Meinberger ◽  
Gabriele Hermes ◽  
Gerhard Sengle ◽  
...  
Keyword(s):  
Vitamin D ◽  
Angiotensin Converting Enzyme ◽  
Receptor Binding ◽  
Viral Entry ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Cell Infection ◽  
S Protein

AbstractObjectiveSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen accountable for the coronavirus disease 2019 (COVID-19) pandemic. Viral entry via binding of the receptor binding domain (RBD) located within the S1 subunit of the SARS-CoV-2 Spike (S) protein to its target receptor angiotensin converting enzyme (ACE) 2 is a key step in cell infection. The efficient transition of the virus is linked to a unique protein called open reading frame (ORF) 8. As SARS-CoV-2 infections can develop into life-threatening lower respiratory syndromes, effective therapy options are urgently needed. Several publications propose vitamin D treatment, although its mode of action against COVID-19 is not fully elucidated. It is speculated that vitamin D’s beneficial effects are mediated by up-regulating LL-37, a well-known antimicrobial peptide with antiviral effects.MethodsRecombinantly expressed SARS-CoV-2 S protein, the extended S1 subunit (S1e), the S2 subunit (S2), the receptor binding domain (RBD), and ORF8 were used for surface plasmon resonance (SPR) studies to investigate LL-37’s ability to bind to SARS-CoV-2 proteins and to localize its binding site within the S protein. Binding competition studies were conducted to confirm an inhibitory action of LL-37 on the attachment of SARS-CoV-2 S protein to its entry receptor ACE2.ResultsWe could show that LL-37 binds to SARS-CoV-2 S protein (LL-37/SStrep KD = 410 nM, LL-37/SHis KD = 410 nM) with the same affinity, as SARS-CoV-2 binds to hACE2 (hACE2/SStrep KD = 370 nM, hACE2/SHis KD = 370 nM). The binding is not restricted to the RBD of the S protein, but rather distributed along the entire length of the protein. Interaction between LL-37 and ORF8 was detected with a KD of 290 nM. Further, inhibition of the binding of SStrep (IC50 = 740 nM), S1e (IC50 = 170 nM), and RBD (IC50 = 130 nM) to hACE2 by LL-37 was demonstrated.ConclusionsWe have revealed a biochemical link between vitamin D, LL-37, and COVID-19 severity. SPR analysis demonstrated that LL-37 binds to SARS-CoV-2 S protein and inhibits binding to its receptor hACE2, and most likely viral entry into the cell. This study supports the prophylactic use of vitamin D to induce LL-37 that protects from SARS-CoV-2 infection, and the therapeutic administration of vitamin D for the treatment of COVID-19 patients. Further, our results provide evidence that the direct use of LL-37 by inhalation and systemic application may reduce the severity of COVID-19.

scholarly journals A new proposed mechanism of some known drugs targeting the SARS-CoV-2 spike glycoprotein using molecular docking

2020 ◽  
Author(s):  
Tarek Moussa ◽  
Nevien Sabry
Keyword(s):  
Receptor Binding ◽  
Protein Interactions ◽  
Viral Entry ◽  
Noncovalent Interactions ◽  
3D Structure ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
S Protein ◽  
Noncovalent Bonds ◽  
Free Energy Of Binding

Abstract COVID-19 is caused by the novel enveloped beta-coronavirus with a genomic RNA closely related to severe acute respiratory syndrome-corona virus (SARS-CoV) and is named coronavirus 2 (SARS-CoV-2). The receptor binding domain (RBD) of the S-protein interacts with the human ACE-2 receptor that enables the initiation of viral entry. Hence, blocking the S-protein interactions by means of synthetic compounds mark the pivotal step for targeting SARS-CoV-2. Most of the six compounds were observed to fit nicely with specific noncovalent interactions, including H bonds, electrostatic, Van der Waals and hydrophobic bonds (pi and sigma bonds). Oseltamivir was found to be the most strongly interacting with the RBD, exhibiting high values of full fitness and low free energy of binding. it formed multiple noncovalent bonds in the region of the active site. Hydroxychloroquine also demonstrated high binding affinity in the solvent accessbility state and fit nicely into the active pocket of the S-protein. The results revealed that these compounds could be potent inhibitors of S-protein that could, to some extent, block its interaction with ACE-2. It is obvious from the 3D structure of SARS-CoV-2 spike protein was changed with the interaction of different drugs, which led to the unsuitability to bind ACE2 receptor. Hence, laboratory studies elucidating the action of these compounds on SARS-CoV-2 are warranted for clinical assessments. Chloroquine, hydroxychloroquine and oseltamivir interacted well with the receptor binding domain of S-protein via noncovalent interactions and recommended as excellent candidates for COVID-19.

scholarly journals Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction

2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Shijulal Nelson-Sathi ◽  
P. K. Umasankar ◽  
E. Sreekumar ◽  
R. Radhakrishnan Nair ◽  
Iype Joseph ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Viral Entry ◽  
In Silico ◽  
Rna Virus ◽  
Binding Domain ◽  
Virus Infectivity ◽  
Receptor Binding Domain ◽  
Synonymous Mutations ◽  
Binding Interface

Abstract Background SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. Results Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. Conclusion Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

scholarly journals Proteolytic Transformation and Stimulation of SARSCov-2 Spike Protein with Human ACE-2 Receptor

2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Sohail S ◽  
◽  
Rana H ◽  
Awan DS ◽  
Sohail F ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Viral Entry ◽  
Attachment Site ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Major Pathway ◽  
Important Relationship

Severe acute respiratory syndrome coronavirus has a great role in causing respiratory illness in humans and has the most important relationship of its spike proteins with host ACE-2 receptors. After entry into the human body, the viral S protein receptor-binding domain binds to human ACE-2 receptor. Two modes explained in this paper of an ACE-2 shedding. The shedding induces the process of viral entry to host cells by binding SARS-CoV-2 proteins. The residues of arginine and lysine in the ACE-2 receptor from 652 to 659 amino acid cleavage by ADAM17 but in TMPRSS2 the residues can be seen on amino acid from 697 to 716. Corona virus genome shows some structural proteins that are responsible for the cellular entry and facilitate the attachment of a virus to the host cell. Virus recognizes the attachment site and binds with it and enter into the cell. Spike protein is split from the cleavage site along its two subunits S1 and S2 then during this process. S2 subunit release RBD (Receptor- Binding Domain) of S1 mediated to the ACE-2. The RBD of S1 consists of 200 amino acid domains. The unknown protein B6ATI which is a neutral amino acid transporter located in ileum is the basic cause for formation of ACE-2 homodimer. In this way S1 domain provides site for another S2 domain. This leads to concealing of the ACE-2 ectodomain cleavage-sites, shedding. It prevents endocytosis of the receptor blocking a major pathway in the viral entry.

scholarly journals The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement

2020 ◽  
Author(s):  
Brian D. Quinlan ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Yan Guo ◽  
Wenhui He ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Neutralizing Antibody ◽  
Binding Domain ◽  
Entry Inhibitor ◽  
Receptor Binding Domain ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Antibody Dependent Enhancement ◽  
Acid Fragment

SUMMARYThe SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates entry of SARS-CoV-2 into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. Antibodies to the RBD domain of SARS-CoV (SARS-CoV-1), a closely related coronavirus which emerged in 2002-2003, have been shown to potently neutralize SARS-CoV-1 S-protein-mediated entry, and the presence of anti-RBD antibodies correlates with neutralization in SARS-CoV-2 convalescent sera. Here we show that immunization with the SARS-CoV-2 RBD elicits a robust neutralizing antibody response in rodents, comparable to 100 µg/ml of ACE2-Ig, a potent SARS-CoV-2 entry inhibitor. Importantly, anti-sera from immunized animals did not mediate antibody-dependent enhancement (ADE) of S-protein-mediated entry under conditions in which Zika virus ADE was readily observed. These data suggest that an RBD-based vaccine for SARS-CoV-2 could be safe and effective.

scholarly journals Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Aluminum Hydroxide Induces Protective Immunity and Reduces Immune Enhancement

2020 ◽  
Author(s):  
Wen-Hsiang Chen ◽  
Xinrong Tao ◽  
Anurodh Agrawal ◽  
Abdullah Algaissi ◽  
Bi-Hung Peng ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Binding Domain ◽  
Pseudotyped Virus ◽  
Receptor Binding Domain ◽  
Protein Vaccine ◽  
S Protein ◽  
Recombinant Receptor ◽  
High Level

AbstractWe developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.

scholarly journals Catechin and curcumin interact with S protein of SARS-CoV2 and ACE2 of human cell membrane: insights from computational studies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Atala B. Jena ◽  
Namrata Kanungo ◽  
Vinayak Nayak ◽  
G. B. N. Chainy ◽  
Jagneshwar Dandapat
Keyword(s):  
Amino Acid ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Computational Study ◽  
Cell Receptor ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Amino Acid Residues ◽  
S Protein

AbstractThe recent outbreak of the coronavirus (SARS-CoV2) is an unprecedented threat to human health and society across the globe. In this context, development of suitable interventions is the need of the hour. The viral spike protein (S Protein) and the cognate host cell receptor ACE2 can be considered as effective and appropriate targets for interventions. It is evident from the present computational study, that catechin and curcumin, not only exhibit strong binding affinity to viral S Protein and host receptor ACE2 but also to their complex (receptor-binding domain (RBD) of the spike protein of SARS-CoV2 and ACE2; RBD/ACE2-complex). The binding affinity values of catechin and curcumin for the S protein, ACE2 and RBD/ACE2-complex are − 10.5 and − 7.9 kcal/mol; − 8.9 and − 7.8 kcal/mol; and − 9.1 and − 7.6 kcal/mol, respectively. Curcumin directly binds to the receptor binding domain (RBD) of viral S Protein. Molecular simulation study over a period of 100 ns further substantiates that such interaction within RBD site of S Protein occurs during 40–100 ns out of 100 ns simulation trajectory. Contrary to this, catechin binds with amino acid residues present near the RBD site of S Protein and causes fluctuation in the amino acid residues of the RBD and its near proximity. Both catechin and curcumin bind the interface of ‘RBD/ACE2-complex’ and intervene in causing fluctuation of the alpha helices and beta-strands of the protein complex. Protein–protein interaction studies in presence of curcumin or catechin also corroborate the above findings suggesting the efficacy of these two polyphenols in hindering the formation of S Protein-ACE2 complex. In conclusion, this computational study for the first time predicts the possibility of above two polyphenols for therapeutic strategy against SARS-CoV2.

scholarly journals A Fungal Defensin Targets the SARS−CoV−2 Spike Receptor−Binding Domain

2021 ◽  
Vol 7 (7) ◽  
pp. 553
Author(s):  
Bin Gao ◽  
Shunyi Zhu
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Single Point ◽  
Binding Domain ◽  
Host Cells ◽  
Single Point Mutation ◽  
Binding Motif ◽  
Microsporum Canis ◽  
Receptor Binding Domain ◽  
Fungal Defensin

Coronavirus Disease 2019 (COVID−19) elicited by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS−CoV−2) is calling for novel targeted drugs. Since the viral entry into host cells depends on specific interactions between the receptor−binding domain (RBD) of the viral Spike protein and the membrane−bound monocarboxypeptidase angiotensin converting enzyme 2 (ACE2), the development of high affinity RBD binders to compete with human ACE2 represents a promising strategy for the design of therapeutics to prevent viral entry. Here, we report the discovery of such a binder and its improvement via a combination of computational and experimental approaches. The binder micasin, a known fungal defensin from the dermatophytic fungus Microsporum canis with antibacterial activity, can dock to the crevice formed by the receptor−binding motif (RBM) of RBD via an extensive shape complementarity interface (855.9 Å2 in area) with numerous hydrophobic and hydrogen−bonding interactions. Using microscale thermophoresis (MST) technique, we confirmed that micasin and its C−terminal γ−core derivative with multiple predicted interacting residues exhibited a low micromolar affinity to RBD. Expanding the interface area of micasin through a single point mutation to 970.5 Å2 accompanying an enhanced hydrogen bond network significantly improved its binding affinity by six−fold. Our work highlights the naturally occurring fungal defensins as an emerging resource that may be suitable for the development into antiviral agents for COVID−19.

ReaxFF-based molecular dynamics simulation of the interaction between plasma ROS and the receptor-binding domain of the spike protein in the capsid protein of SARS-CoV-2

2021 ◽  
Author(s):  
Huichao Wang ◽  
Tong Zhao ◽  
Shuhui Yang ◽  
Liang Zou ◽  
Xiaolong Wang ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Amino Acid ◽  
Capsid Protein ◽  
Receptor Binding ◽  
Hydrogen Abstraction ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Amino Acid Residues ◽  
Global Epidemic

Abstract Under the severe situation of the current global epidemic, researchers have been working hard to find a reliable way to suppress the infection of the virus and prevent the spread of the epidemic. Studies have shown that the recognition and binding of human angiotensin-converting enzyme 2 (ACE2) by the receptor-binding domain (BRD) of spike protein on the surface of SARS-CoV-2 is a crucial step for SARS-CoV-2 to invade human receptor cells, and blocking this process can inhibit the virus from invading human normal cells. Plasma treatment can disrupt the structure of the RBD and effectively block the binding process. However, the mechanism by which plasma blocks the recognition and binding between the two is not clear. In this study, reaction process between reactive oxygen species (ROS) in plasma and the molecular model of RBD was simulated using a reactive molecular dynamics method. The results showed that the destruction of RBD molecule by ROS was triggered by hydrogen abstraction reactions. O and OH abstracted H atoms from RBD, while the H atoms of H2O2 and HO2 were abstracted by RBD. The hydrogen abstraction resulted in the breakage of C-H, N-H, O-H and C=O bonds and the formation of C=C, C=N bonds. The addition reaction of OH increased the number of O-H bonds and caused the formation of C-O, N-O and O-H bonds. The dissociation of N-H bonds led to the destruction of the original structure of peptide bonds and amino acid residues, change the type of amino acid residues, and caused the conversion of N-C and N=C, C=O and C-O. The simulation partially elucidated the microscopic mechanism of the interaction between ROS in plasma and the capsid protein of SARS-CoV-2, providing theoretical support for the control of SARS-CoV-2 infection by plasma, a contribution to overcoming the global epidemic problem.

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