Molecular strategies for antibody binding and escape of SARS-CoV-2 and its mutations

AbstractThe COVID19 pandemic, caused by SARS-CoV-2, has infected more than 200 million people worldwide. Due to the rapid spreading of SARS-CoV-2 and its impact, it is paramount to find effective treatments against it. Human neutralizing antibodies are an effective method to fight viral infection. However, the recent discovery of new strains that substantially change the S-protein sequence has raised concern about vaccines and antibodies’ effectiveness. Here, using molecular simulations, we investigated the binding mechanisms between the S-protein and several antibodies. Multiple mutations were included to understand the strategies for antibody escape in new variants. We found that the combination of mutations K417N, E484K, L452R, and T478K produced higher binding energy to ACE2 than the wild type, suggesting higher efficiency to enter host cells. The mutations’ effect depends on the antibody class. While Class I enhances the binding avidity in the presence of N501Y mutation, class II antibodies showed a sharp decline in the binding affinity. Our simulations suggest that Class I antibodies will remain effective against the new strains. In contrast, Class II antibodies will have less affinity to the S-protein, potentially affecting these antibodies’ efficiency.

Download Full-text

Molecular strategies for antibody binding and escape of SARS-CoV-2 and its mutations

10.1101/2021.03.04.433970 ◽

2021 ◽

Author(s):

Mohamed Hendy ◽

Samuel Kaufman ◽

Mauricio Ponga

Keyword(s):

Neutralizing Antibodies ◽

Class Ii ◽

Host Cells ◽

Class I ◽

Wild Type ◽

Sharp Decline ◽

S Protein ◽

Antibody Class ◽

Binding Avidity ◽

Binding Mechanisms

The COVID19 pandemic, caused by SARS-CoV-2, has infected more than 100 million people worldwide. Due to the rapid spreading of SARS-CoV-2 and its impact, it is paramount to find effective treatments against it. Human neutralizing antibodies are an effective method to fight viral infection. However, the recent discovery of new strains that substantially change the S-protein sequence has raised concern about vaccines and antibodies’ effectiveness. Here, we investigated the binding mechanisms between the S-protein and several antibodies. Multiple mutations were included to understand the strategies for antibody escape in new variants. We found that the combination of mutations K417N and E484K produced higher binding energy to ACE2 than the wild type, suggesting higher efficiency to enter host cells. The mutations’ effect depends on the antibody class. While Class I enhances the binding avidity in the presence of N501Y mutation, class II antibodies showed a sharp decline in the binding affinity. Our simulations suggest that Class I antibodies will remain effective against the new strains. In contrast, Class II antibodies will have less affinity to the S-protein, potentially affecting these antibodies’ efficiency.

Download Full-text

Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

Journal of Virology ◽

10.1128/jvi.79.6.3289-3296.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3289-3296 ◽

Cited By ~ 59

Author(s):

Choong-Tat Keng ◽

Aihua Zhang ◽

Shuo Shen ◽

Kuo-Ming Lip ◽

Burtram C. Fielding ◽

...

Keyword(s):

Amino Acids ◽

Severe Acute Respiratory Syndrome ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Antiviral Agents ◽

Host Cells ◽

Heptad Repeat ◽

Antigenic Determinants ◽

Amino Acid Residues ◽

S Protein

ABSTRACT The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

Download Full-text

Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles

Molecular and Cellular Biology ◽

10.1128/mcb.3.7.1172-1181.1983 ◽

1983 ◽

Vol 3 (7) ◽

pp. 1172-1181

Author(s):

W E Bradley

Keyword(s):

Cell Lines ◽

Chinese Hamster Ovary ◽

High Frequency ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Low Frequency ◽

Class Ii ◽

Class I ◽

Wild Type ◽

Ovary Cells

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.

Download Full-text

Regulation of interference-sensitive crossover distribution ensures crossover assurance in Arabidopsis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2107543118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2107543118

Author(s):

Xiang Li ◽

Jun Zhang ◽

Jiyue Huang ◽

Jing Xu ◽

Zhiyu Chen ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Arabidopsis Thaliana ◽

Mus Musculus ◽

Distribution Patterns ◽

Underlying Mechanism ◽

Class Ii ◽

Class I ◽

Wild Type ◽

Chromosomal Segregation ◽

Normal Class

During meiosis, crossovers (COs) are typically required to ensure faithful chromosomal segregation. Despite the requirement for at least one CO between each pair of chromosomes, closely spaced double COs are usually underrepresented due to a phenomenon called CO interference. Like Mus musculus and Saccharomyces cerevisiae, Arabidopsis thaliana has both interference-sensitive (Class I) and interference-insensitive (Class II) COs. However, the underlying mechanism controlling CO distribution remains largely elusive. Both AtMUS81 and AtFANCD2 promote the formation of Class II CO. Using both AtHEI10 and AtMLH1 immunostaining, two markers of Class I COs, we show that AtFANCD2 but not AtMUS81 is required for normal Class I CO distribution among chromosomes. Depleting AtFANCD2 leads to a CO distribution pattern that is intermediate between that of wild-type and a Poisson distribution. Moreover, in Atfancm, Atfigl1, and Atrmi1 mutants where increased Class II CO frequency has been reported previously, we observe Class I CO distribution patterns that are strikingly similar to Atfancd2. Surprisingly, we found that AtFANCD2 plays opposite roles in regulating CO frequency in Atfancm compared with either in Atfigl1 or Atrmi1. Together, these results reveal that although AtFANCD2, AtFANCM, AtFIGL1, and AtRMI1 regulate Class II CO frequency by distinct mechanisms, they have similar roles in controlling the distribution of Class I COs among chromosomes.

Download Full-text

Acute Myeloid Leukemia (AML) with 9q Aberrations Occuring within a Non-Complex Karyotype Is Highly Associated with CEBPA and NPM1 Mutations - A Joint Analysis of the German-Austrian AML Study Group (AMLSG) and Cancer and Leukemia Group B (CALGB).

Blood ◽

10.1182/blood.v110.11.762.762 ◽

2007 ◽

Vol 110 (11) ◽

pp. 762-762

Author(s):

Andrea Corbacioglu ◽

Stefan Fröhling ◽

Peter Paschka ◽

Guido Marcucci ◽

Andreas Anhalt ◽

...

Keyword(s):

Gene Mutations ◽

Class Ii ◽

Class I ◽

Joint Analysis ◽

Complex Karyotype ◽

Loss Of Function ◽

Wild Type ◽

Npm1 Mutation ◽

Cebpa Mutations ◽

Group 1

Abstract Background: In a previous study we showed that AML with deletion 9q (9q-) occurring in the context of a non-complex karyotype is associated with CEBPA loss-of-function mutations; their prevalence was 41% (Frohling et al., Genes Chromosomes Cancer2005;42:427). We hypothesized that disruption of CEBPA function and loss of a critical segment of 9q cooperate in leukemogenesis. This is consistent with the model of leukemogenesis, in which mutations from different complementation groups cooperate, e.g., CEBPA mutations, representing the class II mutation impairing differentiation, occur simultaneously with 9q- associated with loss and/or mutation of an as yet unidentified gene that confers the proliferative advantage (class I mutation). 9q- is also associated with t(8;21)/RUNX1/RUNX1T1, another class II mutation. Importantly, in initial studies screening for NPM1 mutations, a few patients (pts) with 9q- were reported to harbor NPM1 mutations. Objective: To evaluate the incidence and clinical significance of mutations in the CEBPA and NPM1 genes in a large series of AML with 9q aberrations. Methods: Fifty-seven pts exhibiting a 9q aberration on chromosomal banding analysis were screened for CEBPA and NPM1 mutations. Pts were classified into three groups: those with 9q- occurring as a sole aberration or together with one additional abnormality other than t(8;21) (n=35); pts with 9q- occurring within a complex karyotype, defined as ≥3 abnormalities (n=10); and pts with 9q- secondary to t(8;21) (n=12). Results: The frequencies of CEBPA and NPM1 mutations in group 1 were 49% and 29%, respectively. Strikingly, either a CEBPA or NPM1 mutation was identified in 27 of the 35 (77%) pts within this subgroup. CEBPA and NPM1 mutations did not occur concurrently. In contrast, only one CEBPA and no NPM1 mutations were detected in group 2; no pt in group 3 had mutations in CEBPA or NPM1. Although the number of pts is still limited, within group 1, pts with CEBPA mutations and those with NPM1 mutations had higher CR rates, 86% and 80%, respectively, than pts with wild-type CEBPA and NPM1, whose CR rate was 57%. Overall survival of 9q- pts with CEBPA mutations was significantly better than that of the remaining pts comprising those with NPM1 mutations and pts with wild-type CEBPA and NPM1 (p=0.03). Conclusions: Abnormalities of 9q occurring in AML pts with a non-complex karyotype and without t(8;21) are highly associated with CEBPA or NPM1 gene mutations. CEBPA and NPM1 mutations are mutually exclusive, a finding that further supports the hypothesis that both CEBPA and NPM1 mutations act as class II mutations, which cooperate with a class I mutation affecting a thus far unknown gene on 9q. Currently, additional pts with 9q- are under investigation.

Download Full-text

Xeno-Nucleic Acid (XNA) 2’-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding

Viruses ◽

10.3390/v13101983 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1983

Author(s):

Irani Alves Ferreira-Bravo ◽

Jeffrey J. DeStefano

Keyword(s):

Nucleic Acid ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Dissociation Constants ◽

High Specificity ◽

Binding Domain ◽

Host Cells ◽

Receptor Binding Domain ◽

S Protein ◽

Equilibrium Dissociation Constants

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2ʹ-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10–20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

Download Full-text

ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021671118 ◽

2021 ◽

Vol 118 (14) ◽

pp. e2021671118

Author(s):

Martin G. France ◽

Janina Enderle ◽

Sarah Röhrig ◽

Holger Puchta ◽

F. Chris H. Franklin ◽

...

Keyword(s):

Genetic Analysis ◽

Synaptonemal Complex ◽

Class Ii ◽

Class I ◽

Crossing Over ◽

Wild Type ◽

Homologous Chromosomes ◽

Virtual Absence ◽

Meiotic Progression ◽

Null Mutants

The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5. These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis. Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.

Download Full-text

Computational analyses of the G476S variant of SARS-CoV-2: A focus on the interaction with human ACE-2 and neutralizing antibodies

10.21203/rs.3.rs-98463/v1 ◽

2020 ◽

Author(s):

Alexander Kwarteng ◽

Ebenezer Asiedu ◽

Augustina Angelina Sylverken

Keyword(s):

Structural Dynamics ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Computational Approach ◽

Receptor Binding Domain ◽

Interaction Energies ◽

Wild Type ◽

S Protein ◽

Conformation Changes ◽

Computational Analyses

Abstract Recently, several mutations in the SARS-CoV-2 genome have been identified and reported. However, little is currently known about the influence of these mutations on the infectivity, transmissibility and antigenicity of the virus. Here, using an integrative computational approach, we characterized the G476S variant of SARS-CoV-2 focusing on interactions with ACE-2 and neutralizing antibodies. The substitution of Gly-476 to Ser-476 in the SARS-CoV-2 Receptor-binding domain (RBD) largely affected the structural dynamics of the S-protein leading to significant influence on the interactions with ACE-2 and neutralizing antibodies. Structural properties of the S-protein such as conformation changes, residual fluctuations and residue surface area largely varied between the wild-type and G476S variant, especially in the RBD. Analyses of the interaction energies between S-protein and ACE-2 suggest that the G476S variant may have enhanced interactions with ACE-2 compared to the wild-type. The G476S variant was found to have weaker interactions with the neutralizing antibody H014 compared to the wild-type. Collectively, our findings have implications for the infectivity and antigenicity of the G476S variant of SARS-CoV-2.

Download Full-text

Murine T Lymphocytes Incapable of Producing Macrophage Inhibitory Protein-1 Are Impaired in Causing Graft-Versus-Host Disease Across a Class I But Not Class II Major Histocompatibility Complex Barrier

Blood ◽

10.1182/blood.v93.1.43 ◽

1999 ◽

Vol 93 (1) ◽

pp. 43-50 ◽

Cited By ~ 29

Author(s):

Jonathan S. Serody ◽

Donald N. Cook ◽

Suzanne L. Kirby ◽

Elizabeth Reap ◽

Thomas C. Shea ◽

...

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Graft Versus Host Disease ◽

Class Ii ◽

Class I ◽

Wild Type ◽

Inhibitory Protein ◽

Host Disease ◽

Graft Versus Host

Abstract The routine use of bone marrow transplantation is limited by the occurrence of acute and chronic graft-versus-host disease (GVHD). Current approaches to decreasing the occurrence of GVHD after allogeneic transplantation use T-cell depletion, use immunosuppressive agents, or block costimulatory molecule function. The role of proteins in the recruitment of alloreactive lymphocytes has not been well characterized. Chemokines are a large family of proteins that mediate recruitment of mononuclear cells in vitro and in vivo. To investigate the role of T-cell production of the chemokine macrophage inhibitory protein-1 (MIP-1) in the occurrence of GVHD, splenocytes either from wild-type or from MIP-1−/− mice were administered to class I (B6.C-H2bm1) and class II disparate mice (B6-C-H2bm12). The incidence and severity of GVHD was markedly reduced in bm1 mice receiving splenocytes from MIP-1−/− mice as compared with mice receiving wild-type splenocytes. Bm1 mice receiving MIP-1−/− splenocytes had significantly less weight loss and markedly reduced inflammatory responses in the lung and liver than mice receiving C57BL/6 splenocytes. Bm1 mice receiving MIP-1−/− splenocytes had a markedly decreased production of antichromatin autoantibodies and impaired generation of bm1-specific T lymphocytes versus wild-type mice. However, MIP-1−/− splenocytes easily induced GVHD when administered to bm12 mice. This data show that blockade of chemokine production or function may provide a new approach to the prevention or treatment of GVHD but that chemokines that recruit both CD4+ and CD8+ lymphocytes may need to be targeted.

Download Full-text

Glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

Journal of Virology ◽

10.1128/jvi.01626-21 ◽

2021 ◽

Author(s):

Shijian Zhang ◽

Eden P. Go ◽

Haitao Ding ◽

Saumya Anang ◽

John C. Kappes ◽

...

Keyword(s):

Disulfide Bond ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Etiologic Agent ◽

Host Cells ◽

Wild Type Virus ◽

Virus Infectivity ◽

Wild Type ◽

Spike Glycoprotein ◽

Sugar Addition

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by co-expression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glycoprotein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape, and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vaccines and therapies for COVID-19.

Download Full-text