Patent Grant
11746144
The present disclosure relates to the field of treatment of a COVID-19 infection.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, is a coronavirus closely related to SARS-CoV and Middle East Respiratory Syndrome (MERS) coronaviruses (A. C. Walls et al. (2020)). COVID-19 can manifest in adults as a severe interstitial pneumonia with hyperinflammation while severe respiratory manifestations are rare in children (L. Cristiani et al. (2020); M. Z. Tay, et al. (2020); N. Vabret et al. (2020)). Recently, however, multisystem inflammatory syndrome in children (MIS-C) has been recognized in patients that either tested positive for COVID-19 (by PCR or serology) or had epidemiological links to COVID-19 (S. Riphagen, et al. (2020); L. Verdoni et al. (2020); Z. Belhadjer et al. (2020)). These children present with a constellation of symptoms including hypotension, multiorgan involvement, and elevated inflammatory markers. After initial reports in UK (S. Riphagen, et al. (2020)), many cases have now been reported in Europe (L. Verdoni et al. (2020); Z. Belhadjer et al. (2020)), and New York (USA CDC). However, no such cases have been reported in China, Japan, or South Korea, which have also been severely impacted by the COVID-19 pandemic (ECDC). What is needed are compositions and methods for treating a COVID-19 infection, including MIS-C. The compositions and methods disclosed herein address these and other needs.
Provided herein are methods of treating a COVID-19 infection in a subject, comprising administering to the subject an effective amount of a composition that reduces the superantigen character of SARS-CoV-2 Spike protein. In some embodiments, the compositions are mimetic peptides of the superantigen region. In some embodiments, the compositions are humanized antibodies such as humanized mAb 6D3 that bind to the superantigen region.
Accordingly, provided herein are methods of treating a COVID-19 infection in a subject, comprising administering to the subject an effective amount of one or more of a humanized mAb 6D3, a humanized mAb 14G8, and a functional fragment thereof. In some embodiments, the humanized mAb 6D3 comprises one or more of a VH CDR amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16. In some embodiments, the humanized mAb 6D3 comprises one or more of a VL CDR amino acid sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, the humanized mAb 6D3 comprises (a) a VH domain having an amino acid sequence comprising SEQ ID NO:13, and (b) a VL domain having an amino acid sequence comprising SEQ ID NO:17. In some embodiments, the humanized mAb 14G8 comprises one or more of a VH CDR amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, the humanized mAb 14G8 comprises one or more of a VL CDR amino acid sequence selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42. In some embodiments, the humanized mAb 14G8 comprises (a) a VH domain having an amino acid sequence comprising SEQ ID NO:35, and (b) a VL domain having an amino acid sequence comprising SEQ ID NO:39.
Also included herein are methods of treating a COVID-19 infection in a subject, comprising administering to the subject an effective amount of one or more SARS-CoV-2 superantigenic (SAg) peptides, wherein the one or more peptides comprise SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, the one or more peptides comprise SEQ ID NO:4. In some embodiments, the one or more peptides comprise SEQ ID NO:5. In some embodiments, the one or more peptides comprise SEQ ID NO:6. In some embodiments, the one or more peptides comprise SEQ ID NO:7. In some embodiments, the one or more peptides comprise SEQ ID NO:8. In some embodiments, the one or more peptides comprise SEQ ID NO:9. In some embodiments, the one or more peptides comprise SEQ ID NO:10. In some embodiments, the one or more peptides comprise SEQ ID NO:11. In some embodiments, the one or more peptides comprise SEQ ID NO:12.
In some embodiments of the above-described methods, the subject is a human. The human can be of any age, but in some embodiments, the human is a child and the treatment results in an amelioration of a multisystem inflammatory syndrome. In some embodiments, the treatment results in an amelioration of a pneumonia.
Disclosed herein is the surprising discovery that SARS-CoV-2 encodes a superantigen (SAg) motif near the S1/S2 cleavage site of its Spike protein. This region is highly similar in structure to the SEB SAg motif that interacts with both the TCR and CD28 (G. Arad et al. (2011)) and mediates toxic shock syndrome (TSS). Superantigens are highly potent T cell activators that can bind to MHC class II (MHCII) molecules and/or to TCRs of both CD4+ and CD8+ T cells. The ability of SAgs to bypass the antigen specificity of the TCRs results in broad activation of T cells and a cytokine storm, leading to toxic shock (H. Li, et al. (1999), T. Krakauer (2019)). Notably SAgs do not bind the major (antigenic) peptide binding groove of MHCII, but instead bind other regions as well as the αβTCRs, directly. While early studies showed that bacterial SAgs activate T cells by binding the β-chain of dimeric TCRs at their variable domain (V) (M. T. Scherer, et al. (1993), Y. W. Choi et al. (1989), J. D. Fraser, T. Proft (2008)), more recent studies revealed that they can bind to either α- or β-chains, or both (M. Saline et al. (2010)). As a SAg, SEB enables large-scale T cell activation and proliferation (T. Krakauer (2019)), resulting in massive production of pro-inflammatory cytokines including IFNγ, TNFα and IL-2 from T cells as well as IL-1 and TNFα from antigen presenting cells (APCs) (T. Krakauer (2019)). This cytokine storm leads to multi-organ tissue damage similar to what is now observed in MIS-C.
Accordingly, included in the present invention are methods of treating a COVID-19 infection in a subject, comprising administering to the subject an effective amount of a composition that that prevents the SARS-CoV-2 Spike protein from acting as a SAg. Included herein are compositions and methods for reducing an amount of SARS-CoV-2 Spike protein binding to a T cell receptor, an MHC molecule and/or CD28. In some embodiments, the compositions and methods include or employ a 6D3 antibody, mAb 6D3. In some embodiments, the compositions and methods include or employ a SARS-CoV-2 SAg peptide.
Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicants desire that the following terms be given the particular definition as provided below.
As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
The terms “about” and “approximately” are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
The term “administering” refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
The term “antibody” is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody.
The term “antibody fragment” refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2 and Fv fragments. The phrase “functional fragment or analog” of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example a “functional fragment” refers to a fragment of mAb 6D3 that reduces binding of a SARS-CoV-2 Spike protein SAg region to a T cell receptor (TCR), an MHC molecule and/or CD28. As used herein, “functional fragment” with respect to antibodies, refers to Fv, F(ab) and F(ab′)2 fragments. An “Fv” fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define an target binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site. “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for target binding. The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. F(ab′) fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab′)2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
As used herein, the word “child” refers to a human between the ages of 1 day and 16 years. In some embodiments, the child is between the ages of 1 and 16 years, 3 and 13 years, 5 and 11 years, or 6 and 10 years. In some embodiments, the child is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 years old.
A “composition” is intended to include a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
A “control” is an alternative subject or sample used in an experiment for comparison purpose. A control can be “positive” or “negative.”
“COVID-19” refers herein to a disease or disorder caused in whole or in part by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
As used herein, a “COVID-19 infection symptom” includes, but is not limited to, pneumonia, interstitial pneumonia, interstitial pneumonia with hyperinflammation, multisystem inflammatory syndrome in children (MIS-C), hypotension, sepsis, septic shock, multiple organ disfunction syndrome (MODS), and respiratory failure, dyspnea, respiratory frequency≥30/min, blood oxygen saturation (SpO2)≤93%, PaO2/FiO2 ratio or P/F [the ratio between the blood pressure of the oxygen (partial pressure of oxygen, PaO2) and the percentage of oxygen supplied (fraction of inspired oxygen, FiO2)]<300, and/or lung infiltrates >50% within 24 to 48 hours. In some embodiments, the COVID-19 infection symptom is MIS-C.
“Effective amount” encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, and/or lessen a symptom or sign of a medical condition or disorder (e.g., a COVID-19 infection). Unless dictated otherwise, explicitly or by context, an “effective amount” is not limited to a minimal amount sufficient to reverse, mitigate, and/or lessen a condition. The severity of a disease or disorder, as well as the ability of a treatment to reverse, mitigate, and/or lessen the disease or disorder can be measured, without implying any limitation, by a biomarker or by a clinical parameter. In some instances, an amelioration, reversal, mitigation, and/or lessening is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. The effective amount will vary depending on the compound, such as a mAb 6D3 or COVID-19 SAg peptide, the disorder or conditions and its severity, the route of administration, time of administration, rate of excretion, drug combination, judgment of the treating physician, dosage form, and the age, weight, general health, sex and/or diet of the subject to be treated. In the context of the present methods, an effective amount or dose of a mAb 6D3 antibody or COVID-19 SAg peptide includes an amount that is sufficient to reverse, mitigate, and/or lessen a COVID-19 infection symptom. In some embodiments, an effective amount or dose of a mAb 6D3 or COVID-19 SAg peptide is an amount sufficient to ameliorate a COVID-19 infection symptom. In some embodiments, an effective amount or dose of a mAb 6D3 or COVID-19 SAg peptide is an amount sufficient to ameliorate a pneumonia. In some embodiments, an effective amount or dose of a mAb 6D3 or COVID-19 SAg peptide is an amount sufficient to ameliorate an interstitial pneumonia. In some embodiments, an effective amount or dose of a mAb 6D3 or COVID-19 SAg peptide is an amount sufficient to ameliorate hyperinflammation. In some embodiments, an effective amount or dose of a mAb 6D3 or COVID-19 SAg peptide is an amount sufficient to ameliorate a multisystem inflammatory syndrome in children (MIS-C).
“Homologs” are defined herein as two polynucleotides or two polypeptides that have identity or homology. Homologs include allelic variants, orthologs, and paralogs having the same relevant function (e.g., ability to bind to a COVID-19 SAg or ability to block binding of a COVID-19 SAg to a T cell receptor, a major histocompatibility (MHC) molecule and/or CD28). In some embodiments, homologs have about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92, 91% or 90% homology. In other embodiments, homologs have about 80% or about 85% homology.
“Humanized” forms of non-human (e.g. murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other target-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
The term “identity” or “homology” shall be construed to mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the bases or residues of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity or homology. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) that has a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In one embodiment, default parameters are used for alignment. In one embodiment a BLAST program is used with default parameters. In one embodiment, BLAST programs BLASTN and BLASTP are used with the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.
The term “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature.
The term “monoclonal antibody” or “mAb” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
The term “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.
The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition. In some instances, the terms “treat”, “treating”, “treatment” and grammatical variations thereof, include partially or completely reducing the severity of a COVID-19 infection, partially or completely reducing a multisystem inflammatory syndrome, or partially or completely reducing a pneumonia associated with a COVID-19 infection as compared with prior to treatment of the subject or as compared with the incidence of such symptom in a general or study population.
Compositions and Methods
As noted above, it is disclosed herein that SARS-CoV-2 encodes a superantigen (SAg) motif near the S1/S2 cleavage site of its Spike protein. As used herein, “Spike protein” or “S protein” refers to a polypeptide that mediates binding of a SARS-CoV virus to a cell and/or membrane fusion of the virus to a cell. The Spike protein contains an extracellular domain (EC) with two subunits, a receptor-binding subunit (S1) and a membrane-fusion subunit (S2). S1 contains two domains, an N-terminal domain (S1-NTD) and receptor binding domain (RBD), which play a key role in receptor recognition and binding. During host-virus membrane fusion, Spike protein is usually cleaved at the S1/S2 boundary by host proteases, releasing the spike fusion peptide, which is necessary for virus entry.
In some embodiments, the Spike protein is that identified in a publicly available database as follows: UniProtKB P0DTC2. In some embodiments, the Spike protein comprises the sequence of SEQ ID NO: 1, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1. In some embodiments, the Spike protein is an isoform of SEQ ID NO:1. In some embodiments, the Spike protein is a ortholog of SEQ ID NO:1. The Spike protein of SEQ ID NO: 1 may represent an immature or pre-processed form of mature Spike protein, and accordingly, included herein are mature or processed portions of the Spike protein in SEQ ID NO: 1. In other embodiments, the Spike protein comprises the sequence of SEQ ID NO:1 modified by one or more of the following mutations: D614G, A831V and D839Y/N/E.
Accordingly, included herein are methods of treating a COVID-19 infection in a subject, comprising administering to the subject an effective amount of a composition that that reduces binding of a SARS-CoV-2 Spike protein SAg region to a T cell receptor (TCR), an MHC molecule and/or CD28. In some embodiments, binding to a TCR alpha chain is reduced. In other embodiments, binding to a TCR beta chain is reduced. In some embodiments, the MHC molecule is an MHC Class II. In some embodiments, the reduction in binding is about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% as compared to a control.
One SAg region of the SARS-CoV-2 Spike protein comprises amino acids PRRA (SEQ ID NO:2), which correspond or correlate with amino acid positions 681 to 684 of SEQ ID NO:1. In some embodiments, the SAg region comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids and includes amino acids that correspond or correlate with positions 681 to 684 of SEQ ID NO:1. Another SAg region of the SARS-CoV-2 Spike protein comprises amino acids YNENGTITDAVDCALDPLSETKC (SEQ ID NO:3), which correspond or correlate with amino acid positions 279 to 301 of SEQ ID NO:1.
In some embodiments, the SAg region comprises amino acids TNSPRRAR (SEQ ID NO:4), which correspond or correlate with amino acid positions 678 to 685 of SEQ ID NO:1. In some embodiments, the SAg region comprises amino acids YQTQTNSPRRAR (SEQ ID NO:5), which correspond or correlate with amino acid positions 674 to 685 of SEQ ID NO:1. In some embodiments, the SAg region comprises amino acids PRRARS (SEQ ID NO:6), which correspond or correlate with amino acid positions 681 to 686 of SEQ ID NO:1. In some embodiments, the SAg region comprises amino acids PRRASVASQ (SEQ ID NO:7), which correspond or correlate with amino acid positions 681 to 690 of SEQ ID NO:1. In some embodiments, the SAg region comprises amino acids PRRASVASQSI (SEQ ID NO:8), which correspond or correlate with amino acid positions 681 to 692 of SEQ ID NO:1. In some embodiments, the SAg region comprises amino acids TNSPRRASVASQ (SEQ ID NO:9), which correspond or correlate with amino acid positions 678 to 690 of SEQ ID NO:1. In some embodiments, the SAg region comprises amino acids QTNSPRRARSVAS (SEQ ID NO:10), which correspond or correlate with amino acid positions 677 to 689 of SEQ ID NO:1. In some embodiments, the SAg region comprises amino acids ECDIPIGAGICASYQTQTNSPRRARSV (SEQ ID NO:11), which correspond or correlate with amino acid positions 661 to 687 of SEQ ID NO:1. In some embodiments, the SAg region comprises amino acids ECDIPIGAGICASYQTQTNSPRRAR (SEQ ID NO:12), which correspond or correlate with amino acid positions 661 to 685 of SEQ ID NO: 1. In some embodiments, the SAg region comprises amino acids YNENGTITDAVDCALDPLSETKC (SEQ ID NO:3), which correspond or correlate with amino acid positions 279 to 301 of SEQ ID NO:1.
The present invention also includes compositions such as peptide mimetics of the SARS-CoV-2 SAg region and methods for using those compositions in treating a COVID-19 infection in a subject. As used herein, the term “peptide mimetic” refers to an amino acid sequence that comprises or corresponds to a SARS-CoV-2 Spike protein SAg region. Accordingly, included herein are SARS-CoV-2 SAg peptides comprising amino acids PRRA (SEQ ID NO:2), which correspond or correlate with amino acid positions 681 to 684 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 SAg peptide comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids and includes amino acids that correspond or correlate with positions 681 to 684 of SEQ ID NO:1.
In some embodiments, the SARS-CoV-2 SAg peptide comprises amino acids TNSPRRAR (SEQ ID NO:4), which correspond or correlate with amino acid positions 678 to 685 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 SAg peptide comprises amino acids YQTQTNSPRRAR (SEQ ID NO:5), which correspond or correlate with amino acid positions 674 to 685 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 SAg peptide comprises amino acids PRRARS (SEQ ID NO:6), which correspond or correlate with amino acid positions 681 to 686 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 SAg peptide comprises amino acids PRRASVASQ (SEQ ID NO:7), which correspond or correlate with amino acid positions 681 to 690 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 SAg peptide comprises amino acids PRRASVASQSI (SEQ ID NO:8), which correspond or correlate with amino acid positions 681 to 692 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 SAg peptide comprises amino acids TNSPRRASVASQ (SEQ ID NO:9), which correspond or correlate with amino acid positions 678 to 690 of SEQ ID NO:1. In some embodiments, the SAg peptide comprises amino acids QTNSPRRARSVAS (SEQ ID NO:10), which correspond or correlate with amino acid positions 677 to 689 of SEQ ID NO:1. In some embodiments, the SAg peptide comprises amino acids ECDIPIGAGICASYQTQTNSPRRARSV (SEQ ID NO:11), which correspond or correlate with amino acid positions 661 to 687 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 SAg peptide comprises amino acids ECDIPIGAGICASYQTQTNSPRRAR (SEQ ID NO:12), which correspond or correlate with amino acid positions 661 to 685 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 SAg peptide comprises amino acids YNENGTITDAVDCALDPLSETKC (SEQ ID NO:3), which correspond or correlate with amino acid positions 279 to 301 of SEQ ID NO:1.
It should be understood that the present invention includes methods of administering an effective amount of one or more SARS-CoV-2 SAg peptides for treating a COVID-19 infection in a subject. Provided are methods of treating a COVID-19 infection in a subject, comprising administering to the subject an effective amount of one or more SARS-CoV-2 superantigenic (SAg) peptides, wherein the one or more peptides are selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:2. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:3. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:4. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:5. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:6. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:7. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:8. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:9. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:10. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:11. In some embodiments, the method of treating a COVID-19 infection in a subject, comprises administering to the subject an effective amount of a peptide comprising SEQ ID NO:12.
Also included in the present invention are methods of treating a COVID-19 infection in a subject, comprising administering to the subject an effective amount of one or more of an mAb 6D3, an mAb 20B1, an mAb 14G8, a functional fragment thereof, or a humanized form thereof. These methods can be used separately or in conjunction with the methods of administering one or more SARS-CoV-2 SAg peptides also described herein.
The term “mAb 6D3” refers to a 6D3 antibody as described in Patent Application Publication US 2014/0234325 (U.S. application Ser. No. 14/346,981) and/or Patent Application Publication US 2016/0039914 (U.S. application Ser. No. 14/774,283), each of which is incorporated by reference herein in its entirety. In some embodiments, the mAb 6D3 comprises a VH amino acid sequence of SEQ ID NO:13. In some embodiments, the mAb 6D3 comprises a VL amino acid sequence of SEQ ID NO:17. In some embodiments, the mAb 6D3 comprises a CDR amino acid sequence of one or more of SEQ ID NO:14 (VH CDR1), SEQ ID NO:15 (VH CDR2) and SEQ ID NO:16 (VH CDR3). In some embodiments, the mAb 6D3 comprises a CDR amino acid sequence of one or more of SEQ ID NO:18 (VL CDR1), SEQ ID NO:19 (VL CDR2) and SEQ ID NO:20 (VL CDR3). In some embodiments, the mAb 6D3 comprises a CDR3 amino acid sequence of SEQ ID NO:16 and a CDR3 amino acid sequence of SEQ ID NO:20.
In some embodiments, the mAb 6D3 is humanized. A “humanized mAb 6D3” refers to a chimeric antibody that comprises two or more CDR amino acid sequences of an mAb 6D3 and one or more human antibody sequences. In some embodiments, the humanized mAb 6D3 comprises human framework regions. In some embodiments, the humanized mAb 6D3 further comprises a human Fc region. In some embodiments, the humanized mAb 6D3 comprises two or more murine CDRs selected from the group consisting of SEQ ID NO:14 (VH CDR1), SEQ ID NO:15 (VH CDR2), SEQ ID NO:16 (VH CDR3), SEQ ID NO:18 (VL CDR1), SEQ ID NO:19 (VL CDR2) and SEQ ID NO:20 (VL CDR3). In some embodiments, the humanized mAb 6D3 comprises three or more murine CDRs selected from the group consisting of SEQ ID NO:14 (VH CDR1), SEQ ID NO:15 (VH CDR2), SEQ ID NO:16 (VH CDR3), SEQ ID NO:18 (VL CDR1), SEQ ID NO:19 (VL CDR2) and SEQ ID NO:20 (VL CDR3). In some embodiments, the humanized mAb 6D3 comprises four or more murine CDRs selected from the group consisting of SEQ ID NO:14 (VH CDR1), SEQ ID NO:15 (VH CDR2), SEQ ID NO:16 (VH CDR3), SEQ ID NO:18 (VL CDR1), SEQ ID NO:19 (VL CDR2) and SEQ ID NO:20 (VL CDR3). In some embodiments, the humanized mAb 6D3 comprises five or more murine CDRs selected from the group consisting of SEQ ID NO:14 (VH CDR1), SEQ ID NO:15 (VH CDR2), SEQ ID NO:16 (VH CDR3), SEQ ID NO:18 (VL CDR1), SEQ ID NO:19 (VL CDR2) and SEQ ID NO:20 (VL CDR3). In some embodiments, the humanized mAb 6D3 comprises all of SEQ ID NO:14 (VH CDR1), SEQ ID NO:15 (VH CDR2), SEQ ID NO:16 (VH CDR3), SEQ ID NO:18 (VL CDR1), SEQ ID NO:19 (VL CDR2) and SEQ ID NO:20 (VL CDR3).
The term “mAb 20B1” refers to a 20B1 antibody as described in Patent Application Publication US 2014/0234325 (U.S. application Ser. No. 14/346,981) and/or Patent Application Publication US 2016/0039914 (U.S. application Ser. No. 14/774,283). In some embodiments, the mAb 20B1 comprises a VH amino acid sequence of SEQ ID NO:21. In some embodiments, the mAb 20B1 comprises a VL amino acid sequence of SEQ ID NO:25. In some embodiments, the mAb 20B1 comprises a CDR amino acid sequence of one or more of SEQ ID NO:22 or SEQ ID NO:29 (VH CDR1), SEQ ID NO:23 or SEQ ID NO:30 (VH CDR2) and SEQ ID NO:24 or SEQ ID NO:31 (VH CDR3). In some embodiments, the mAb 20B1 comprises a CDR amino acid sequence of one or more of SEQ ID NO:26 or SEQ ID NO:32 (VL CDR1), SEQ ID NO:27 or SEQ ID NO:33 (VL CDR2) and SEQ ID NO:28 or SEQ ID NO:34 (VL CDR3). In some embodiments, the mAb 20B1 comprises a CDR3 amino acid sequence of SEQ ID NO:24 or SEQ ID NO:31 and a CDR3 amino acid sequence of SEQ ID NO:28 or SEQ ID NO:34.
In some embodiments, the mAb 20B1 is humanized. A “humanized mAb 20B1” refers to a chimeric antibody that comprises two or more CDR amino acid sequences of an mAb 20B1 and one or more human antibody sequences. In some embodiments, the humanized mAb 20B1 comprises human framework regions. In some embodiments, the humanized mAb 20B1 further comprises a human Fc region. In some embodiments, the humanized mAb 20B1 comprises two or more murine CDRs selected from the group consisting of SEQ ID NO:22 or SEQ ID NO:29 (VH CDR1), SEQ ID NO:23 or SEQ ID NO:30 (VH CDR2), SEQ ID NO:24 or SEQ ID NO:31 (VH CDR3), SEQ ID NO:26 or SEQ ID NO:32 (VL CDR1), SEQ ID NO:27 or SEQ ID NO:33 (VL CDR2) and SEQ ID NO:28 or SEQ ID NO:34 (VL CDR3). In some embodiments, the humanized mAb 20B1 comprises three or more murine CDRs selected from the group consisting of SEQ ID NO:22 or SEQ ID NO:29 (VH CDR1), SEQ ID NO:23 or SEQ ID NO:30 (VH CDR2), SEQ ID NO:24 or SEQ ID NO:31 (VH CDR3), SEQ ID NO:26 or SEQ ID NO:32 (VL CDR1), SEQ ID NO:27 or SEQ ID NO:33 (VL CDR2) and SEQ ID NO:28 or SEQ ID NO:34 (VL CDR3). In some embodiments, the humanized mAb 20B1 comprises four or more murine CDRs selected from the group consisting of SEQ ID NO:22 or SEQ ID NO:29 (VH CDR1), SEQ ID NO:23 or SEQ ID NO:30 (VH CDR2), SEQ ID NO:24 or SEQ ID NO:31 (VH CDR3), SEQ ID NO:26 or SEQ ID NO:32 (VL CDR1), SEQ ID NO:27 or SEQ ID NO:33 (VL CDR2) and SEQ ID NO:28 or SEQ ID NO:34 (VL CDR3). In some embodiments, the humanized mAb 20B1 comprises five or more murine CDRs selected from the group consisting of SEQ ID NO:22 or SEQ ID NO:29 (VH CDR1), SEQ ID NO:23 or SEQ ID NO:30 (VH CDR2), SEQ ID NO:24 or SEQ ID NO:31 (VH CDR3), SEQ ID NO:26 or SEQ ID NO:32 (VL CDR1), SEQ ID NO:27 or SEQ ID NO:33 (VL CDR2) and SEQ ID NO:28 or SEQ ID NO:34 (VL CDR3). In some embodiments, the humanized mAb 20B1 comprises six murine CDRs selected from the group consisting of SEQ ID NO:22 or SEQ ID NO:29 (VH CDR1), SEQ ID NO:23 or SEQ ID NO:30 (VH CDR2), SEQ ID NO:24 or SEQ ID NO:31 (VH CDR3), SEQ ID NO:26 or SEQ ID NO:32 (VL CDR1), SEQ ID NO:27 or SEQ ID NO:33 (VL CDR2) and SEQ ID NO:28 or SEQ ID NO:34 (VL CDR3).
The term “mAb 14G8” refers to a 14G8 antibody as described in Patent Application Publication US 2014/0234325 (U.S. application Ser. No. 14/346,981) and/or Patent Application Publication US 2016/0039914 (U.S. application Ser. No. 14/774,283). In some embodiments, the mAb 14G8 comprises a VH amino acid sequence of SEQ ID NO:35. In some embodiments, the mAb 14G8 comprises a VL amino acid sequence of SEQ ID NO:39. In some embodiments, the mAb 14G8 comprises a CDR amino acid sequence of one or more of SEQ ID NO:36 (VH CDR1), SEQ ID NO:37 (VH CDR2) and SEQ ID NO:38 (VH CDR3). In some embodiments, the mAb 14G8 comprises a CDR amino acid sequence of one or more of SEQ ID NO:40 (VL CDR1), SEQ ID NO:41 (VL CDR2) and SEQ ID NO:42 (VL CDR3). In some embodiments, the mAb 14G8 comprises a CDR3 amino acid sequence of SEQ ID NO:38 and a CDR3 amino acid sequence of SEQ ID NO:42.
In some embodiments, the mAb 14G8 is humanized. A “humanized mAb 14G8” refers to a chimeric antibody that comprises two or more CDR amino acid sequences of an mAb 14G8 and one or more human antibody sequences. In some embodiments, the humanized mAb 14G8 comprises human framework regions. In some embodiments, the humanized mAb 14G8 further comprises a human Fc region. In some embodiments, the humanized mAb 14G8 comprises two or more murine CDRs selected from the group consisting of SEQ ID NO:36 (VH CDR1), SEQ ID NO:37 (VH CDR2), SEQ ID NO:38 (VH CDR3), SEQ ID NO:40 (VL CDR1), SEQ ID NO:41 (VL CDR2), and SEQ ID NO:42 (VL CDR3). In some embodiments, the humanized mAb 14G8 comprises three or more murine CDRs selected from the group consisting of SEQ ID NO:36 (VH CDR1), SEQ ID NO:37 (VH CDR2), SEQ ID NO:38 (VH CDR3), SEQ ID NO:40 (VL CDR1), SEQ ID NO:41 (VL CDR2), and SEQ ID NO:42 (VL CDR3). In some embodiments, the humanized mAb 14G8 comprises four or more murine CDRs selected from the group consisting of SEQ ID NO:36 (VH CDR1), SEQ ID NO:37 (VH CDR2), SEQ ID NO:38 (VH CDR3), SEQ ID NO:40 (VL CDR1), SEQ ID NO:41 (VL CDR2), and SEQ ID NO:42 (VL CDR3). In some embodiments, the humanized mAb 14G8 comprises five or more murine CDRs selected from the group consisting of SEQ ID NO:36 (VH CDR1), SEQ ID NO:37 (VH CDR2), SEQ ID NO:38 (VH CDR3), SEQ ID NO:40 (VL CDR1), SEQ ID NO:41 (VL CDR2), and SEQ ID NO:42 (VL CDR3). In some embodiments, the humanized mAb 14G8 comprises all of SEQ ID NO:36 (VH CDR1), SEQ ID NO:37 (VH CDR2), SEQ ID NO:38 (VH CDR3), SEQ ID NO:40 (VL CDR1), SEQ ID NO:41 (VL CDR2), and SEQ ID NO:42 (VL CDR3).
In one aspect, the disclosed methods can be employed 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years; 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 months; 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours prior to the onset of a COVID-19 symptom; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 or more days; 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 years after the onset of COVID-19 symptom. In some embodiments, the disclosed methods can be employed prior to or following the administering of another anti-SARS-CoV-2 agent.
A SARS-CoV-2 SAg peptide and/or a humanized mAb 6D3 described herein can be administered to the subject via any route including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
Dosing frequency for a SARS-CoV-2 SAg peptide and/or a humanized mAb 6D3 of any preceding aspects, includes, but is not limited to, at least once every year, once every two years, once every three years, once every four years, once every five years, once every six years, once every seven years, once every eight years, once every nine years, once every ten year, at least once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, at least once every month, once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, daily, twice a day, three times a day, four times a day, or five times a day. Administration can also be continuous and adjusted to maintaining a level of the compound within any desired and specified range.
The following examples are set forth below to illustrate the compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Viruses. SARS-CoV-2 (PODTC2) and SARS-CoV (CVHSA P59594) spike models were generated using SWISS-MODEL (L. Bordoli, T. Schwede, (Springer, 2011)), based on the resolved spike glycoprotein structures of SARS-CoV-2 (D. Wrapp et al. (2020)) (PDB: 6VSB) and SARS-CoV (W. Song, et al. (2018)) (PDB: 6ACD). The missing loops in the crystal structures were built using libraries of backbone fragments (Y. Zhang, J. Skolnick, (2005)) or by constraint space de novo reconstruction of these backbone segments (M. C. Peitsch (1995)). Two mutants associated with European Covid-19 patients (B. Korber et al. (2020)) were constructed using CHARMM-GUI (S. Jo, T. Kim, et al. (2008)): one is the main strain mutant D614G and the other contains four mutations including Q239K, A831V, D614G and D839Y. These two SARS-CoV-2 spike mutants together with the SARS-CoV-2 (PODTC2) originally taken from Wuhan were used to investigate the binding to αβTCR, and MHCII (PDB: 2XN9) (M. Saline et al. (2010)) using ClusPro (D. Kozakov et al. (2017)) and PRODIGY (L. C. Xue, et al. (2016)).
Generation of a binary complex between SARS-CoV-2 spike and T cell receptor (TCR). SARS-CoV-2 spike model in the prefusion state was generated using SwissModel (Bordoli, L. & Schwede, T. (Springer, 2011)) based on the resolved cryo-EM structure (Protein Data Bank (PDB): 6VSB (Wrapp, D. et al. (2020)) for the spike glycoprotein where one of the receptor binding domains (RBDs) is in the up conformation. The structure of the T cell receptor (TCR) containing both TCRα and TCRβ chains was taken from the crystal structure of the ternary complex between human TCR, staphylococcal enterotoxin H (SEH) and human major histocompatibility complex class II (MHCII) molecule (Saline, M. et al. (2010)). Using protein-protein docking software ClusPro (Kozakov, D. et al. (2017)), a series of binary complexes were constructed in silico for SARS-CoV-2 spike and TCR. 30 clusters of conformations were obtained for spike-TCR binary complexes, upon clustering 1000 models generated by ClusPro. The clusters were rank-ordered by cluster size, as recommended (Kozakov, D. et al. (2017)). All models were analyzed which found that the majority (>90%) showed that TCR bound to spike via its constant domain. Given that the constant domain is proximal to the cell membrane and TCR employs the variable domain for binding superantigens and/or antigen/MHC complexes (Saline, M. et al. (2010)), restraints were then added to the docking simulations to prevent the binding of TCR constant domain and filter out those conformers where the variable domain would bind to the spike. This led to 27 clusters (based on a set of 666 models) from ClusPro. Interestingly, 45% of models showed the binding of TCR near the region of “PRRA” insert and 46% of models showed the binding of TCR within multiple RBDs. Thus, two hot spots were identified for TCR binding within SARS-CoV-2 spike: one is near “PRRA” insert and the other within the RBD. Representative members belonging to the top-ranking clusters are presented in
SARS-CoV-2 spike model in the prefusion state was generated using SwissModel (A. Waterhouse et al. (2018)) based on the resolved cryo-EM structure (Protein Data Bank (PDB): 6VSB) (Wrapp, D. et al. (2020)) for the S glycoprotein where one of the receptor binding domains (RBDs) is in the up conformation and the other two in the down conformation. The structure of the T cell receptor (TCR) containing both α- and β-chains was taken from the crystal structure (PDB: 2XN9) of the ternary complex resolved for human TCR, staphylococcal enterotoxin H (SEH) and human major histocompatibility complex class II (MHCII) molecule (Saline, M. et al. (2010)). Using protein-protein docking software ClusPro (Kozakov, D. et al. (2017)), a series of binary complexes were constructed in silico for SARS-CoV-2 spike and TCR. 30 clusters of conformations were obtained for spike-TCR binary complexes, upon clustering the 1000 models generated by ClusPro. The clusters were rank-ordered by cluster size, as recommended (Kozakov, D. et al. (2017)). We analyzed all models and found that a large fraction showed that TCR bound to spike via its constant domain. Given that the constant domain is proximal to the cell membrane and TCR employs the variable domain for binding superantigens (SAgs) and/or antigen/MHC complexes (Saline, M. et al. (2010)), restraints to the docking simulations were then added to filter out those conformers where the variable domain would bind to the spike. This led to 27 clusters (based on a set of 666 models) from ClusPro. Interestingly, in 45% of the generated models, the TCR was observed to bind to a spike epitope that contained the “PRRA” insert; and in 46% of models we observed an interaction between the TCR and one or two of the three RBDs.
Thus, two hot spots were identified for TCR binding within the SARS-CoV-2 spike: one overlapping with the “PRRA” insert and the other on the RBD surface. Representative members belonging to the top-ranking clusters are presented in
Generation of a binary complex between SARS-CoV spike and TCR. Further, SARS-CoV (SARS1) spike model in the prefusion state was generated using SwissModel (A. Waterhouse et al. (2018)) based on the cryo-EM structure resolved for SARS-CoV spike (PDB: 6ACD) (W. Song, et al. (2018)) where one of the RBDs is in the up conformation, and the other two in the down conformation. Following the same approach as we did for SARS-CoV-2 spike, a series of binary complexes were constructed in silico for SARS-CoV spike and TCR using ClusPro (Kozakov, D. et al. (2017)). Using the same filtering procedure, this led to 30 clusters (based on 686 models), among which 38% showed the binding of TCR to multiple RBDs (see
Generation of a binary complex between MERS-CoV spike and TCR. MERS-CoV spike model was generated using SwissModel (A. Waterhouse et al. (2018)) based on the cryo-EM structure resolved for MERS-CoV spike (W. Song, et al. (2018)) (PDB: 5X5F) in which one of RBDs is in the up conformation. 30 clusters (based on 588 models) were predicted by ClusPro (Kozakov, D. et al. (2017)). 56% of models led to TCR binding to the RBDs. Two representative poses from these most populated clusters are shown in
Examination of neurotoxin-like and other bioactive segments on SARS-CoV-2 spike.
Generation of a ternary complex between SARS-CoV-2 spike, TCR, and MHCII. Structure of the human MHCII was taken from the crystal structure of the ternary complex (Saline, M. et al. (2010)) (PDB: 2XN9) between human TCR, SEH and MHCII. First, docking simulations were performed to generate binary complexes between MHCII and SARS-CoV-2 spike. Six representative MHCII-spike binary complexes were selected to explore further docking of TCR to form a ternary complex. All predicted ternary complex models of MHCII-Spike-TCR were analyzed. Tertiary MHCII-Spike-TCR complex models were selected following three filtering criteria: (1) TCR either binds near “PRRA” insert region or the RBD; (2) the binding regions involve homologous superantigen or toxin binding motifs predicted for SARS-CoV (
In silico mutagenesis of D839 of SARS-CoV-2 spike. D839 of the SARS-CoV-2 spike were mutated in silico to asparagine, glutamic acid and tyrosine in line with the aforementioned mutant D839Y/N/E observed in a new strain from Europe. To this aim, PyMOL mutagenesis tool (DeLano, W. L. (2002)) was used and the change in local conformation and energetics were evaluated in the complex formed with TCR. The most probable rotamers were selected and energetically minimized in the presence of the bound TCR (conformation shown in
Analysis of NGS immunosequencing data from COVID-19 patients. Blood collection from 38 patients (42 samples) with mild/moderate COVID-19, and 8 patients (24 samples) with severe/hyperinflammatory COVID-19 was performed under institutional review board approval number 2020-039. The patients and controls, and their immune repertoires, were part of a previously published cohort (Schultheiss C, et al. (2020)). For details of NGS data acquisition, please refer to the earlier work (Schultheiss C, et al. (2020)). Only productive TRB rearrangements were used and all repertoires were normalized to 20,000 reads. For the analyses, we used R version 3.5.1 for plotting of TRBV and TRBJ gene usage as previously described (Simnica D, et al. (2019), Simnica D, et al. (2019)). Differences in principal component analysis were studied by Pillai-Bartlett test of MANOVA. To study TRBJ gene diversity, J genes were extracted if they were part of rearrangements containing TRBV rearrangements expanded in patients with hyperinflammatory COVID-19. Frequencies of J gene families were summarized per repertoire and plotted separately for each rearrangement. See
Generation of complexes between SARS-CoV-2 spike, SAg-specific TCRs and MHC II. Four TCR Vβ genes (TRBV5-6, TRBV14, TRBV13 and TRBV24-1) were found to be overrepresented in severe/hyperinflammatory COVID-19 patients (
MIS-C manifests as persistent fever and hyperinflammation with multi organ system involvement including cardiac, gastrointestinal, renal, hematologic, dermatologic and neurologic symptoms (S. Riphagen, et al. (2020); L. Verdoni et al. (2020); Z. Belhadjer et al. (2020)) which are highly reminiscent of toxic shock syndrome (TSS) (D. E. Low (2013), A. Cook, et al. (2020)) (Table 1), rather than Kawasaki disease due to marked demographic, clinical, and laboratory differences (L. Verdoni et al. (2020)). The similarities to TSS and the association of MIS-C with COVID-19 indicate that SARS-CoV-2 can possess superantigenic fragments that induce an inflammatory cascade and contribute to the hyperinflammation and cytokine storm features observed in severe adult COVID-19 cases (M. Z. Tay, et al. (2020), N. Vabret et al. (2020)). The question is: does SARS-CoV-2 S possess superantigenic fragments that can elicit such reactions upon binding proteins involved in the host cell cytotoxic adaptive immune response? Such a reaction was not observed in the SARS-CoV pandemic of 2003 (shortly SARS1). What is unique to SARS-CoV-2, and how recent mutations in SARS-CoV-2 S promotes such an increased virulence?
ataken from refs (S. Riphagen, et al. (2020); L. Verdoni et al. (2020); Z. Belhadjer et al. (2020));
btaken from refs (8-11).
TSS can be caused by two types of superantigens (SAgs): bacterial or viral. Bacterial SAgs have been broadly studied. They include proteins secreted by Staphylococcus aureus and Streptococcus pyogenes that induce inflammatory cytokine gene induction and toxic shock. Typical examples are TSS toxin 1 (TSST1), and staphylococcal enterotoxins B (SEB) and H (SEH). They are highly potent T cell activators that can bind to MHC class II (MHCII) molecules and/or to TCRs of both CD4+ and CD8+ T cells. The ability of SAgs to bypass the antigen specificity of the TCRs results in broad activation of T cells and a cytokine storm, leading to toxic shock (H. Li, et al. (1999), T. Krakauer (2019)). Notably SAgs do not bind the major (antigenic) peptide binding groove of MHCII, but instead bind other regions as well as the αβTCRs, directly. While early studies showed that bacterial SAgs activate T cells by binding the β-chain of dimeric TCRs at their variable domain (V) (M. T. Scherer, et al. (1993), Y. W. Choi et al. (1989), J. D. Fraser, T. Proft (2008)), more recent studies revealed that they can bind to either α- or β-chains, or both (M. Saline et al. (2010)). The question is then, does SARS-CoV-2 S possess any superantigenic fragments/domains that can bind to the αβTCRs?
Here, computational modelling was used to determine whether the SARS-CoV-2 S possesses SAg activity. An insert present in SARS-CoV-2 S was found, which is absent from SARS1 and MERS, mediates high affinity, non-specific binding to the TCR. Notably, a motif of ˜20 amino acids enclosing this insert unique to SARS-CoV-2 among beta coronaviruses has sequence and structure features highly similar to those of the SEB toxin. Furthermore, this analysis shows that a SARS-CoV-2 S mutation detected in a European strain can enhance TCR binding, indicating such mutations can account for geographical differences in MIS-C occurrence.
First, whether SARS-CoV-2 S binds to the αβTCR was examined. To this aim, a SARS-CoV-2 S structural model was constructed based on the cryo-EM structure resolved for the spike glycoprotein (D. Wrapp et al. (2020)), and the X-ray structure of αβTCR resolved in a ternary complex was used with SEH and MHCII (M. Saline et al. (2010)), and a series of structural models were generated for possible SARS-CoV-2 S glycoprotein—TCR complex formation using ClusPro (D. Kozakov et al. (2017)). These simulations revealed two most probable TCR binding sites on each monomer of the S trimer: one on the receptor binding domain (RBD; residues R319-K529), and the other near the S1/S2 cleavage site between the subunits S1 and S2. The former was also shared by SARS1 and MERS-CoV S, while the latter was unique (or strongly preferred) in SARS-CoV-2 S, as de-scribed in detail in the
The TCRVβ-binding epitope on SARS-CoV-2 S is centered around a sequence motif, P681RRA684 (SEQ ID NO:2) (or shortly PRRA, hereafter), and its sequential and spatial neighbors. Comparison of SARS-CoV-2 S to other beta-coronavirus spike protein sequences showed (20) that SARS-CoV-2 is distinguished by the existence of this four-residue insertion, PRRA, preceding the furin cleavage site (R685-5686 peptide bond) between the subunits S1 and S2 of each protomer (
Notably the exposure of this motif and its close sequential neighbors is further accentuated in the S1 trimeric form (
The insertion PRRA (SEQ ID NO:2) together with the sequentially preceding seven amino acids and succeeding Arg (fully conserved among β-coronaviruses) have been pointed out to form a motif, Y674QTQTNSPRRAR685 (SEQ ID NO:48), homologous to that of neurotoxins from Ophiophagus (cobra) and Bungarus genera, as well as neurotoxin-like regions from three RABV strains (J. P. Changeux, et al. (2020)) (
This close sequence similarity to both bacterial and viral SAgs, in support of the potential superantigenic character of the amino acid stretch Y674-R685 of SARS-CoV-2 S led to further analyze its local sequence and structural properties. This analysis led to an interesting sequence similarity between the partially overlapping fragment T678-Q690 of the spike and the SEB superantigenic peptide Y150NKKKATVQELD161 (SEQ ID NO:112) (
Significantly, the structures of the two peptides exhibit a remarkable similarity (
This analysis overall indicates that the segment T678NSPRRAR685 (SEQ ID NO:4) forms a putatively superantigenic core, consistently aligned against various bacterial or viral SAgs (
The SEB superantigen peptide Y150NKKKATVQELD161 (SEQ ID NO:112) has been reported to bind CD28 (G. Arad et al. (2011)), a T cell receptor that provides co-stimulatory signals required for T cell activation and survival. CD28 and TCRV domains share the same (immunoglobulin) fold (
Finally, because of the homologous superantigenic segment of SEB binding CD28, the potential binding of SARS-CoV-2 spike E661-R685 onto CD28 was also tested, considering the possibility that the target of SARS2 spike superantigenic segment is CD28. The simulations indicated that the same segment can equally bind to CD28, further supporting the strong propensity of the fragment to stimulate T cell activation.
The existence of potential superantigenic, toxic or intercellular-adhesion molecule (ICAM)-like sequence fragments in SARS1 was thoroughly examined by Li et al. following the 2003 pandemic (Y. Li et al. (2004)). This led to the identification of the nine sequence stretches including three Botulinum neurotoxin type D or G precursors, and two motifs that have a high similarity with the intercellular adhesion molecule 1 (ICAM-1). Comparative analysis with SARS-CoV-2 spike sequence revealed that seven of these sequence motifs are conserved between SARS-CoV and SARS-CoV-2 (with >68% sequence identity) (
ICAM-1 involvement is critical to mediating immune and inflammatory responses. The observed interaction of the ICAM-1-like motif of SARS-CoV-2 S with TCRVα, in tandem with the interaction of the above discussed putative SAg motif (around the insert PRRA) with TCRVα, is to further strengthen the association of the virus with the T cell and the ensuing activation. Precisely, N280-E281-N282 and T286 belonging to the ICAM-like fragment closely interact with the TCRVα CDRs; mainly T286 (spike) makes close contacts with S94 (CDR3), E281 (spike) forms a hydrogen bond with T51 (CDR2), and N280 and N282 (spike) closely associate with R69 (
The SARS-CoV-2 spike binding region harbors three residues that have been recently reported to have mutated in new strains from Europe and USA (S. H. Zhan, et al. (2020), B. Korber et al. (2020)): D614G, A831V and D839Y/N/E). The former two may potentially interact with MHCII; while the latter (D839, European strain) is located close to TCRVβ and strongly interacts with N30; (
Further examination of the SARS-CoV-2 S segments sequentially homologous to the neurotoxinlike sequences identified (S. H. Zhan, et al. (2020)) for SARS1 S (rows highlighted in green in
A recent study (Mateus J, et al. (2020)) detected significant T cell reactivity against 66 epitopes on the SARS-CoV-2 S glycoprotein in people who have not been exposed to the virus, inviting attention to memory response acquired upon exposure to human CoVs (HCoVs) such as common cold HCoV-OC43, -HKU1, -NL63, and -229E, which share sequence homology with SARS-CoV-2 genome. A total of 142 such cross-reactive epitopes were identified upon screening 474 peptides in the SARS-CoV-2 proteome (Mateus J, et al. (2020)).
The next experiment examined whether the neurotoxin-like regions identified here were among these cross-reactive epitopes. Notably, of the top-ranking four epitopes (ranked by T cell reactivity measured by spot-forming cells (SFC)/106 PBMCs), two (peptides 321-335 and 316-330) belong to the neurotoxin-like fragment T299-Y351, and one (236-250) to 234-262. In fact, the former was completely spanned by eight partially overlapping cross-reactive epitopes as illustrated in the
Among the 66 epitopes, we note 661-675, which lies within the SAg-like region E661-R685 (
The SARS-CoV-2 S binding region harbors three residues that have been recently reported to have mutated in new strains from Europe and USA (Korber B, et al. (2020), Zhan S H, Deverman B E, & Chan Y A (2020)): D614G, A831V and D839Y/N/E. The former two may potentially interact with MHCII based on a ternary model we generated for SARS-CoV-2 S, MHCII and TCR (
SAg binding to specific TCR Vβ chains results in Vβ skewing, such that T cells with specific Vβ chains and diverse antigen specificity dominate the TCR repertoire(Li H, Llera A, Malchiodi E L, & Mariuzza R A (1999), Scherer M T (1993)). If the motif identified in SARS-CoV-2 S acts as a SAg, it can be reasoned that patients with mild/moderate COVID-19 disease courses and recovery without hyperinflammation show adaptive immune responses mediated by T cells recognizing SARS-CoV-2 epitopes in a CDR3-mediated fashion; whereas patients with severe/hyperinflammatory COVID-19 would show immune responses consistent with at least partial SAg recognition. NGS immunosequencing data from 38 patients (42 samples) with mild/moderate COVID-19 and 8 patients (24 samples) with severe, hyperinflammatory COVID-19, which were part of a previously studied cohort (Schultheiss C, et al. (2020)). Principal component analysis (PCA) of the TCR β-chain variable gene (TRBV) repertoires corresponding to the two groups revealed that patients with mild/moderate COVID-19 course clustered apart from those with severe/hyperinflammatory COVID-19 (
Differential gene usage analysis showed that several TRBV genes were overrepresented in the severe/hyperinflammatory COVID-19 patient group (
Together, these results show that patients with severe and hyperinflammatory COVID-19 show expansion of TCRs using distinct V genes, along with J gene/CDR3 diversity in these rearrangements, compatible with a SAg selection process.
Finally, next experiment studied structurally resolved TCRs that contained Vβ chains encoded by the genes TRBV5-6, TRBV13, TRBV14 and TRBV24-1 enriched in severe/hyperinflammatory COVID-19 patients. Whether these TCRs could bind the SAg-like region E661-R685 of the SARS-CoV-2 S similarly to the TCR in
An understanding of the immunopathology leading to severe manifestations of COVID-19, in both adults and children, is of critical importance for effective management and treatment of the disease. MIS-C shows remarkable similarity to pediatric TSS (S. Riphagen, et al. (2020), L. Verdoni et al. (2020), Z. Belhadjer et al., (2020), D. E. Low, (2013), A. Cook, et al. (2020)). Using in silico modeling and analysis, it was found that SARS-CoV-2 encodes a superantigen motif near its S1/S2 cleavage site. This region is highly similar in structure to the SEB SAg motif that interacts with both the TCR and CD28 (G. Arad et al. (2011)) and mediates TSS. SEB enables large-scale T cell activation and proliferation, resulting in massive production of pro-inflammatory cytokines including IFNγ, TNFα and IL-2 from T cells as well as IL-1 and TNFα from APCs (T. Krakauer (2019)). This cytokine storm leads to multi-organ tissue damage similar to what is now observed in MIS-C. We therefore propose that MIS-C observed in COVID-19 patients may be mediated by superantigen activity of the SARS-CoV-2 S protein. Furthermore, these findings show that the hyperinflammation observed in severe cases of COVID-19 in adults can also be driven by the SAg-like activity of the S protein. Indeed, SAgs induce an inflammatory cytokine signature similar to that which predicts severity and death in COVID-19, including IL-6, TNFα, IL-8 and IL-10 (Krakauer T (2019), Del Valle D M, et al. (2020)). Moreover, the analysis of the T cell immune response in COVID-19 patients shows that those with more severe and hyperinflammatory clinical courses exhibit TCRVβ skewing consistent with SAg activity.
To date, MIS-C is mostly observed in Europe and East Coast of North America, and has not been described in Asia, despite sizeable outbreaks of COVID-19 (S. Riphagen, et al. (2020); L. Verdoni et al. (2020); Z. Belhadjer et al. (2020)) (CDC and ECDC). It is shown herein that a mutation at D839 found in a European strain of SARS-CoV-2 enhances the binding affinity of the SAg motif to the TCR. This can explain the geographical skewing of MIS-C to areas where the European strain is endemic, and identification of other strain-specific mutations helps predict where future outbreak of MIS-C may occur.
A study of SARS1 immunogenicity, conducted with a cohort of 128 individuals who have recovered from SARS1 (Li C K, et al. (2008)), showed that the SARS1 spike 18-mer D649-L666 (DIPIGAGICASYHTVSLL, SEQ ID NO:113) is one of the peptides most frequently recognized by T cells, among the screened 1,843 peptides that span the whole SARS1 CoV proteome (Table III in Li et al (Li C K, et al. (2008))). This segment coincides with the SARS1 S region E647-R667 that is sequentially (and structurally) homologous to our SARS-CoV-2 spike SAg-like motif E661-R685 (
These findings indicate that immunomodulatory therapeutic options used for TSS can also be effective for MIS-C, including IVIG and steroids. Given structural similarities between SEB and the S protein SAg motif, cross-reactivity of these immunoglobins explains that at least in part the response of MIS-C cases to IVIG. Other FDA-approved anti-inflammatory drugs tested in models of SEB TSS can also be effective, including CTLA4-Ig which can inhibit CD28 co-stimulation (S. J. Whitfield et al. (2017)), and the mTOR inhibitor rapamycin (T. Krakauer, et al. (2010)), which is already in use for COVID-19. In addition, humanized monoclonal anti-SEB Abs have been described (E. A. Larkin, et al. (2010)) that can also be of therapeutic benefit in MIS-C patients. Notably, it has been shown in the mouse model of TSS that lethal SEB superantigen challenge can be prevented by short peptide mimetics of its superantigen motif (G. Arad et al. (2011)). Short peptide mimetics of SARS-CoV-2 spike superantigen region can be employed to prevent/attenuate inflammatory cytokine gene induction and toxic shock in MIS-C patients.
At present, the majority of antibody therapies under investigation are designed to target the SARS-CoV-2 receptor binding domains (RBDs) (M. Yuan et al., (2020), X. Chi et al. (2020)), and the simulations also indicated that RBD might potentially interact with TCRs. However, compared with RBDs, relatively fewer mutations are found in the SAg region of SARS-CoV-2; notably, the “PRRA” insert is unique to SARS-CoV-2 and retained among all of its isolates sequenced to date (S. H. Zhan, et al. (2020), B. Korber et al., (2020)). It is constructive to design antibodies or drugs targeting this SAg region, to not only block the cleavage essential to enabling viral entry (A. C. Walls et al. (2020), M. Hoffmann et al. (2020)) and modulate the SAg-induced inflammatory cytokine gene induction (T. Krakauer (2019)), but also block the cleavage essential to enabling viral entry (Walls A C, et al. (2020), Hoffmann M, et al. (2020)). Alternatively, combination therapies that target both the SAg-like region and the RBD can prove useful.
Fortunately, severe respiratory manifestations of COVID-19 in children as well as development of MIS-C are rare. This is due to trained immunity (L. Cristiani et al. (2020)) or cross-viral immunity to other coronavirus strains (A. Grifoni, et al. (2020)). T and B cells play an important role in the anti-viral response. CD4+ and CD8+ T cells from convalescent COVID-19 patients can recognize a range of SARS2 epitopes, and the S protein represents a major target (A. Grifoni, et al. (2020)). T cells from unexposed individuals can also respond to S protein epitopes from SARS-CoV-2, which supports the hypothesis of cross-viral immunity from other coronavirus strains (A. Grifoni, et al. (2020)). However, why only a fraction of infected children develop MIS-C is unclear. The present study shows that the mutation D839Y found in a European strain of SARS-CoV-2 enhances the binding affinity of the SAg motif to the TCR. This can explain the geographical skewing of MIS-C to areas where the European strain is endemic. A poor initial antibody response to the virus fails to neutralize the SAg, as recently shown in MIS-C patients (Weisberg S P, et al. (2020)), leading to immune enhancement following re-exposure. Certain HLA types are more permissive of binding SAg, and indeed HLA has been shown to play a role in COVID-19 susceptibility (Nguyen A, et al. (2020)). Of the nine cases initially reported in the UK, six were of Afro-Caribbean descent, which also suggests a potential genetic component to susceptibility (Riphagen S, et al. (2020)). In addition, approximately 80% of individuals over age 12 harbor anti-SEB antibodies (LeClaire R D & Bavari S (2001), McGann V G, Rollins J B, & Mason D W (1971)), which may provide protection against the SAg effects of SARS-CoV-2 S protein. The prevalence of preexisting anti-SEB antibodies can also contribute to the age distribution of severe COVID-19 cases in adults, as protective SEB titers fall in older adults after age 70.
Approximately a third or fewer of MIS-C patients tested positive for the SARS-CoV-2, but the majority (but not all) have serologic evidence of infection or a history of exposure to COVID-19 (S. Riphagen, et al. (2020); L. Verdoni et al. (2020); Z. Belhadjer et al. (2020)). This indicates that the SARS-CoV-2 SAg causes a delayed hyperinflammation response in certain children. SAgs have been implicated in autoimmunity by triggering self-reactive T cells (H. Li, et al. (1999)). Antibody-mediated enhancement upon re-exposure to the virus can also contribute to uncontrolled infection and inflammation (S. M. C. Tirado, K.-J. Yoon (2003)). Despite a negative nasopharyngeal PCR test, the virus can still be present in the gastrointestinal tract (Y. Xu et al. (2020)). MIS-C patients demonstrate unusually severe GI symptoms, abdominal pain, vomiting and diarrhea, in addition to severe myocardial dysfunction and cardiac shock (S. Riphagen, et al. (2020); L. Verdoni et al. (2020); Z. Belhadjer et al. (2020)) and such severe GI symptoms are also frequently associated with the SAg (A. Cook, et al. (2020)). In the case of SEB, cleavage and release of a specific fragment is responsible for induction of GI symptoms. The SARS-CoV-2 SAg-like structure shown herein can be similarly cleaved and underlie the GI symptoms observed in MIS-C patients.
It was also observed that a neurotoxin-like segment (T299-Y351) partially overlapping with the RBD exhibited a high affinity to bind TCRs. Notably, this region was recently observed to elicit strong and frequent T cell reactivity mediated by CD4+ T cells in donors who have not been exposed to SARS-CoV-2 (Mateus J, et al. (2020)). This invites attention to its ability to trigger neurotoxic immune response in individuals who have not been exposed to CoVs that contain sequentially homologous peptides.
In summary, disclosed herein are five major observations: (a) PRRAR and sequential neighbors interact with TCRVβ residues D56, R70 and E74 at the CDRs, and this association closely resembles that of SEB SAg with TCRVβ; (b) nearby D839 participates in this interaction and its mutation to tyrosine further strengthens the association with TCRVβ; (c) a sequence motif (N280-T286) typical of ICAM-1 further interacts with the TCRVα further stabilizing or enhancing the association between the viral spike and host cell TCR; and (d) a neurotoxin-like motif (T299-Y351) shows a high tendency to bind TCRs and trigger neurotoxic responses. This latter effect can be attenuated if the SARS-CoV-2-infected individual has been exposed HCoVs that contain homologous segments, as suggested (Mateus J, et al. (2020)) by a recent study; and (e) adult patients with severe/hyperinflammatory COVID-19 exhibit a skewed TCR Vβ repertoire distinguishing them from patients with mild/moderate COVID-19. Overall, these results from both computational modeling and NGS immunosequencing of TCRBs analysis of human samples indicate that strategies used for the treatment of SEB-mediated TSS or approaches to block the interaction of the S protein with TCRs can help reduce hyperinflammatory manifestations or (neuro)toxic effects of COVID-19 in both adults and children.
Introduciton
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause severe interstitial pneumonia with hyperinflammation (Tay et al., 2020; Vabret et al., 2020), as well as many extrapulmonary manifestations (Gupta et al., 2020). A novel multisystem inflammatory syndrome (MIS), reported in both children (MIS-C) and adults (MIS-A), has been observed in patients that either tested positive for, or had epidemiological links to, COVID-19 (Belhadjer et al.; Cheung et al., 2020; Riphagen et al.; Verdoni et al.). MIS-C manifests as persistent fever and hyperinflammation with multi-organ involvement (Belhadjer et al.; Cheung et al., 2020; Riphagen et al.; Verdoni et al.). The clinical similarity between MIS-C/A and the toxic shock syndrome (TSS) caused by bacterial superantigens (SAgs) led to the hypothesis that SARS-CoV-2 might possess a SAg-like motif that triggers hyperinflammation (Cheng et al., 2020; Noval Rivas et al., 2020). Comparison with bacterial toxins indeed revealed a motif in the SARS-CoV-2 spike (S) protein, the sequence and structure of which highly resemble a segment of a bacterial SAg, staphylococcal enterotoxin B (SEB). SAg-like character of the S protein was further supported by T cell receptor (TCR) skewing typical of reaction to SAgs, which was observed in severe COVID-19 patients (Cheng et al., 2020).
The location of the SAg-like motif in the S protein is worthy of attention. SARS-CoV-2 S is a homotrimer, belonging to the family of human coronaviruses (HCoVs), which includes SARS-CoV and Middle East Respiratory Syndrome (MERS), as well as common cold HCoVs NL63, 229E, OC43 and HKU1 (Coutard et al., 2020; Cui et al., 2019; Forni et al., 2017). Each HCoV protomer is composed of two subunits, S1 and S2, playing different roles in viral infection. S1 contains the receptor-binding domain (RBD) that binds to the host cell receptor (human angiotensin converting enzyme 2 (ACE2) for SARS-CoV-2, SARS-CoV, and HCoV-NL63) (Benton et al., 2020; Hoffmann et al., 2020; Matsuyama et al., 2020; Shang et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Yan et al., 2020); whereas S2 contains the fusion peptide required for viral entry (Coutard et al., 2020; Cui et al., 2019; Forni et al., 2017). The SAg-like motif (residues E661-R685) lies at the C-terminus of S1 (Cheng et al., 2020), at the boundary with S2. Membrane fusion requires two successive cleavages by host cell proteases, one at the S1/S2 interface (peptide bond R68515686), and the other at S2′ (R81515816) (Coutard et al., 2020; Hoffmann et al., 2020; Matsuyama et al., 2020; Shang et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Yan et al., 2020). Thus, the SAg-like region overlaps with the S1/S2 cleavage site of the S protein (
Another feature at the SAg-like region is a unique insertion, 681PRRA684 (SEQ ID NO:2), immediately neighboring the cleavage site R685↑5686 (
This polybasic site, 681PRRAR685 (SEQ ID NO:114), can thus serve as a target for SARS-CoV-2 S-neutralizing antibodies (Abs). Most SARS-CoV-2 S Abs under investigation target the RBD (and some, the N-terminal domain, NTD) (Cao et al., 2020b; Chi et al., 2020; Hansen et al., 2020; Pinto et al., 2020; Renn et al., 2020; Shi et al., 2020; Yuan et al., 2020).
The present study focuses here on this polybasic site as a target for mAb binding. The recently detected sequence- and structure-similarity between the PRRA-insert-enclosing SAg-like motif and the bacterial toxin SEB indicated that previously generated anti-SEB monoclonal Abs (mAbs) can bind the viral SAg-like motif, and in particular the segment 682RRAR685 (SEQ ID NO:2), and can thus block access to the S1/S2 cleavage site. The in silico examination of the possible interactions of known anti-SEB mAbs (Dutta et al., 2015) with SARS-CoV-2 S revealed that SEB-specific mAb 6D3 has a high affinity for binding to the S1/S2 site. The models further show that the 6D3 binding site overlaps with those of TMPRSS2 and/or furin, indicating that 6D3 can impede viral entry. Experiments conducted with live viruses confirmed that 6D3 inhibited viral entry. Given that its binding site does not overlap with those of known Abs (
Results
Anti-SEB antibody 6D3 is distinguished by its high affinity to bind SARS-CoV-2 S SAg-like region. As shown in the recent work (Cheng et al., 2020), the S residues E661-R685 that enclose the polybasic segment 681PRRAR685 (SEQ ID NO:114) are sequentially and structurally similar to the segment T150-D161 of SEB. Given this strong similarity, it was examined if mAbs specific for SEB (Dutta et al., 2015; Varshney et al., 2011) can neutralize SARS-CoV-2 S. The close proximity (or adjacency) of the SAg-like region to the cleavage bond R685↑S686 further indicated that an anti-SEB mAb that cross-reacts with SARS-CoV-2 can have the added potential to block the cleavage site essential to viral entry, apart from its ability to attenuate the SAg-mediated hyperinflammatory cytokine storm (Krakauer, 2019).
Three SEB-specific mAbs, 14G8, 6D3, and 20B1, have been generated as effective blockers of the SAg activity of SEB in an animal model of TSS (Varshney et al., 2011). Examination of their crystal structures shows that these mAbs bind different sites on SEB (Dutta et al., 2015), as illustrated in
Among these three SEB mAbs, 6D3 was the only one able to bind to the SARS-CoV-2 S SAg motif (
Among those 6D3-interacting S residues, N603 has been identified as an N-linked glycan site by site-specific glycan analysis of SARS-CoV-2 S (Watanabe et al., 2020) (
These results indicated that 6D3 can decrease the exposure of the cleavage site to the extracellular environment and interfere with SARS-CoV-2 viral entry upon competing with the host cell proteases TMPRSS2 and furin whose binding to the cleavage site, is essential to S protein priming for viral entry. Next, the investigation of the neutralizing effect of 6D3 was tested in live virus experiments.
Anti-SEB antibody, 6D3, inhibits SARS-CoV-2 infection in live virus assays. Here, whether the SEB-specific mAb 6D3 possessed any neutralizing efficacy vis-à-vis SARS-CoV-2 viral entry was investigated. To this end, the ability of 6D3 to inhibit SARS-CoV-2 infection was tested in an in vitro cell culture infection system. Antibodies were incubated with SARS-CoV-2 for 1 hour and then added to plated Vero-E6 cells. At 48 hours post infection, viral infection was analyzed by immunofluorescence using antibodies against dsRNA or SARS-CoV-2 S protein. (
These results indicate that 6D3, can also block viral entry in a concentration-dependent manner, in addition to its high affinity binding to the SARS-CoV2 superantigen-like motif and potentially blocking its interaction with TCRs. Toward assessing whether 6D3 competitively binds the S1/S2 site in the presence of the proteases, the spike-binding mechanisms and affinities of TMPRSS2 and furin was explored and presented next.
TMPRSS2 and/or furin bind to the S1/S2 site in close association with the PRRA (SEQ ID NO:2) insert. The protease-binding characteristics of the S1/S2 site were analyzed to assess whether Abs that targets the PRRA (SEQ ID NO:2) site can also hinder the access of proteases. The S1/S2 site, also known as furin-cleavage site, typically contains eight central residues including the polybasic segment (here 680SPRRAR↑SV687, SEQ ID NO:115), flanked by solvent-accessible residues on both sides (Tian et al., 2012). The resulting structural models generated for the interactions of TMPRSS2 and furin with the S protein are presented in the respective
TMPRSS2 catalytic residues (H296, D345 and 5441) were observed to bind near 681PRRARS686 (SEQ ID NO:6) in 7.5% of the generated models (
In the case of furin binding, 70% of the structural models showed the catalytic residues (D153, H194 and 5368) stabilized in close proximity of 681PRRARS686 (SEQ ID NO:6) (see
Overall, the analysis shows that TMPRSS2 or furin engage in tight intermolecular interactions, in which the basic residues R682 and R683 reach out to the catalytic site of either protease. Binding of either enzyme is accommodated by changes in the local conformations near the cleavage region. However, this analysis also shows that furin binds with higher potency and probability, compared to TMPRSS2. Most importantly, 6D3 and the proteases compete for the same binding site (
An acidic residue cluster at VII CDR2 is the hallmark of Abs targeting the furin-like cleavage site. The study pointed to the distinctive ability 6D3 to bind to the S1/S2 cleavage site while other mAbs (in Table 4) did not show such a binding propensity. Which sequence/structure features distinguish 6D3 from others was investigated. Abs target viruses mainly through their three complementarity determining regions (CDR1-3) in the variable domains, especially in the heavy chains (Li et al., 2020).
A poly-acidic CDR2 at the VH chain thus emerges as a hallmark of the mAbs that target the polybasic furin-like cleavage site. As shown in
As shown in
Discussion
A new strategy for combatting SARS-CoV-2: repurposing of antibodies that target the S1/S2 cleavage site. SARS-CoV-2 S is the main determinant of cell entry and the major target of neutralizing Abs (Cao et al., 2020b; Chi et al., 2020; Hansen et al., 2020; Pinto et al., 2020; Renn et al., 2020; Shi et al., 2020; Yuan et al., 2020). The majority of COVID-19 Ab therapies under investigation are designed to target the S protein RBD, while other potential neutralizing epitopes have also been found (Cao et al., 2020b; Chi et al., 2020; Hansen et al., 2020; Liu et al., 2020; Pinto et al., 2020; Renn et al., 2020; Shi et al., 2020; Yuan et al., 2020). Given the high glycosylation and antigenic variability of SARS-CoV-2 S (Graham et al., 2019), a combination of mAbs that target multiple sites and multiple conformations of SARS-CoV-2 S, is likely the most effective strategy. Besides blocking ACE2 binding, distinct neutralizing mechanisms have been proposed, including Ab-dependent cell cytotoxicity and phagocytosis (Pinto et al., 2020) and restraining the structural changes of SARS-CoV-2 Spike (Chi et al., 2020).
Proteolytic cleavage of SARS-CoV-2 S is the second critical step, succeeding ACE2 binding, in the life cycle of SARS-CoV-2. TMPRSS2 and furin inhibitors have been found to block cell entry of SARS-CoV-2 (Bestle et al., 2020; Hoffmann et al., 2020). The critical role of the furin-cleavage site in SARS-CoV-2 infectivity and Ab activity is also demonstrated in a recent study where the deletion ΔPRRA reduced SARS-CoV-2 viral replication in a human respiratory and attenuated infectivity (Johnson et al., 2021). Unlike TMPRSS2, furin is a ubiquitous proprotein convertase and is required for normal development and function (Thomas, 2002) and its inhibition is not a viable strategy. But, design or repurposing of Abs that block the S1/S2 site is an attractive alternative solution that avoids effects on the (other) activities of TMPRSS2 and furin.
It is well known that the SARS-CoV-2 spike is heavily glycosylated, and the possible interference of glycans with Ab binding is a plausible consideration (Casalino et al., 2020). Notably, 6D3-binding did not give rise to steric clash with the N-linked glycan sequons near the S1/S2 site (e.g. N603, or N657N658 as reported (Watanabe et al., 2020)). In addition SARS-CoV-2 S was predicted to be 0-glycosylated at 5673, T678 and 5686 near the S1/S2 cleavage site (Andersen et al., 2020), yet to be confirmed by experiments (Shajahan et al., 2020; Watanabe et al., 2020). 6D3 can target directly the S1/S2 site of SARS-CoV-2 S (as the host proteases do) without any shielding effect by glycans. On the contrary, the glycosylation near N603 can even assist in promoting its binding near the PRRA (SEQ ID NO:2) site. The modulating role of glycans on the structure and dynamics of the S glycoprotein has been pointed out to be a feature that could be harnessed in anti-SARS-CoV-2 vaccine development (Casalino et al., 2020).
The ability of the polybasic insert to bind antibodies may have escaped prior cryo-EM studies with mutant S protein. It has been a challenge to resolve The S1/S2 loop in cryo-EM studies of HCoV S proteins. First, pre-activation of HCoV S during protein preparation results in a mixture of cleaved and uncleaved spikes (Cai et al., 2020). Second, local conformational changes near the S1/S2 region may differ between cleaved and intact structures, as observed in influenza viruses (Steinhauer, 1999). Third, multiple conformations, if not a disordered state, may exist near that region, as indicated by microseconds simulations and ab initio modeling (Lemmin et al., 2020). Therefore, most of cryo-EM studies of SARS-CoV-2 S protein complexed with Abs have resorted to variants where the 682RRAR685 (SEQ ID NO:121) segment has been replaced by GSAS or SGAG (Barnes et al., 2020; Cao et al., 2020b; Chi et al., 2020; Liu et al., 2020; Lv et al., 2020b; Pinto et al., 2020; Zhou et al., 2020a; Zost et al., 2020) (Table 4). These ‘mutant spikes’ may have precluded the discovery of binding of Abs to the S1/S2 site. Molecular modeling and simulations provided insights into the interactions at this region, including those with proteases and other receptors (Cheng et al., 2020; Jaimes et al., 2020; Lemmin et al., 2020). Such modeling studies was utilized (
6D3 is a repurposable anti-SEB mAb that targets the S1/S2 site and inhibits viral infection. 6D3 is an Ab originally discovered for neutralizing the superantigenic bacterial toxin SEB. it's the present study indicates its use as repurposable mAb against SARS-CoV-2 S protein, by virtue of its ability to bind a sequence motif shared between SEB and S protein. The recent study revealed the high similarity between SARS-CoV-2 S amino acids E661-R685 and SEB amino acids T150-D161, which can contribute to hyperinflammation and MIS-C/A pathogenesis through a SAg-induced immune activation (Cheng et al., 2020). This hypothesis was supported by the clinical and laboratory features observed in MIS-C and severe COVID-19 patients, which were similar to those of toxic shock syndrome (TSS) caused by bacterial toxins such as SEB (Cheng et al., 2020; Noval Rivas et al., 2020). Adult patients with severe Covid-19 (Cheng et al., 2020) as well as children with the multisystem inflammatory syndrome (MIS-C) (Porritt et al., 2020) displayed TCR skewing typical of SAg-induced immune responses. Among the three mAbs discovered against SEB, 6D3 was the only one specific to the region of interest (
Another feature was the fact that this SAg-like segment (that binds 6D3) overlapped with the furin-like cleavage site characteristic of SARS-CoV-2 (and MERS and HCOVs HKU1 and OC43; see
By binding the viral spike protein, SARS-CoV-2 specific antibodies in the blood or mucosal surface could prevent the virus from binding to and infecting target cells. The antibody neutralization assay that we performed in cell culture simulates this scenario, where the specific mAb 6D3 incubated with SARS-CoV-2 binds and neutralizes the virus's ability to attach to the cell receptor and to initiate infection in vitro. Thus, mAb 6D3 can have a differentiating dual role in not only inhibiting viral entry but also blocking the SARS-CoV-2 superantigen-like motif-induced T cell activation, cytokine storm, and hyperinflammation. The next experimental steps assessing the in vivo effect of mAb 6D3 in relevant mouse models of SARS-CoV-2 infection are currently underway.
mAbs with a cluster of acidic residues at their VII CDR2 can mitigate viral infections caused by CoVs that contain furin-like cleavage sites. HCoVs include three highly pathogenic viruses, SARS-CoV-2, SARS-CoV and MERS, and four circulating endemic viruses (HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1) which cause mild to moderate upper respiratory diseases (Coutard et al., 2020; Cui et al., 2019; Forni et al., 2017). Many individuals who have not been exposed to SARS-CoV-2 possess SARS-CoV-2 Spike reactive T cells, due to cross-reaction of immune responses generated against other HCoV strains (Grifoni et al., 2020; Mateus et al., 2020). Cross-reactive antibodies between human βCoV strains have also been identified, including those between SARS and SARS-CoV-2 (Huang et al., 2020; Lv et al., 2020a). Indeed, SARS monoclonal antibody 5309 can potently neutralize both SARS and SARS-CoV-2(Pinto et al., 2020). Furthermore, the effectiveness of IVIG (Belhadjer et al.; Riphagen et al.; Verdoni et al.), may, in part, be due to the presence of cross-reactive antibodies against other HCoV stains. These findings show designing wide spectrum Abs with cross-reactivity among HCoVs. The two Abs (6D3 and 4A8) identified in this study to present the suitable paratope for binding the PRRAR or similar polybasic inserts can block the S1/S2 cleavage site in HCoVs that encode furin-like cleavage sites (
Alternative strategies targeting the S1/S2 site in the light of these repurposable mAbs. Based on the scaffold of 6D3 heavy chain, mini-proteins can be designed to target SARS-CoV-2, MERS, HCoV-OC43 or HKU1, to block CoV entry. Notably, designed de novo mini-proteins have been shown to block ACE2 binding, based on the scaffold of ACE2 (Cao et al., 2020a). Very recently, neuropilin-1 (NRP1) has been identified as a host factor for SARS-CoV-2 infection, bound to the 681RRAR685 (SEQ ID NO:121) segment (Daly et al., 2020). Remarkably, blockade of this interaction by RNAi or mAb against NRP1 significantly reduced in vitro SARS-CoV-2 cellular entry (Cantuti-Castelvetri et al., 2020; Daly et al., 2020). 6D3 can block the binding of NRP1. At present, no clinical treatments or prevention strategies are available for HCoVs (Cui et al., 2019). The present work leads to an improved understanding of coronavirus immunity, facilitating future studies to understand mechanisms of antibody recognition and neutralization, and help screen SARS-CoV-2 Abs for treatment of COVID-19. These findings also show designing therapeutic approaches using a combination of 6D3 and known neutralizing mAbs that bind the RBD, for treating severe COVID-19 and MIS-C/A patients and/or combatting the spread of the newly emerging variants.
Method Details
In vitro viral inhibition assays. SARS-CoV-2 viral assays were performed in UCLA BSL3 high containment facility, following previous procedure(Garcia et al., 2020). SARS-CoV-2 Isolate USA-WA1/2020 was obtained from BEI Resources of National Institute of Allergy and Infectious Diseases (NIAID). Mouse Fab 6D3 (IgG2b) was generated as(Varshney et al., 2011). Vero-E6 cells were plated in 96-well plates (5×103 cells/well). 6D3 IgG2b or mouse IgG2b isotype control (Bio X Cell) were incubated with virus (100 PFU/well) for 1 hour at room temperature prior to addition to Vero-E6 cells. After 48 hours post-infection the cells were fixed with methanol for 30-60 minutes in −20° C. Cells were washed 3 times with PBS and permeabilized using blocking buffer (0.3% Triton X-100, 2% BSA, 5% Goat Serum, 5% Donkey Serum in 1×PBS) for 1 hour at room temperature. Subsequently, cells were incubated with mouse anti-dsRNA antibody (Absolute Antibody, 1:200) or anti-SARS-CoV-2 spike antibody (Sino Biological, 1:200) at 4° C. overnight. Cells were then washed 3 times with PBS and incubated with fluorescence conjugated secondary antibody: Goat anti-mouse IgG Secondary Antibody, Alexa Fluor 555 (Fisher Scientific, 1:1000) for 1 hour at room temperature. Nuclei were stained with DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride) (Life Technologies) at a dilution of 1:5000 in PBS for 10 minutes. Cells were analyzed by fluorescence microscopy. Images were obtained using a Biorevo BZ-X710 (Keyence) microscope and software.
Structural data for SARS-CoV-2, human TMPRSS2 and furin. SARS-CoV-2 (residues A27-D1146; UniProt ID: P0DTC2) spike models were generated using SWISS-MODEL (Waterhouse et al., 2018), based on the resolved SARS-CoV-2 Spike glycoprotein structures of SARS-CoV-2 in different conformational states (PDBs: 6VSB (Wrapp et al., 2020) and 6VXX (Walls et al., 2020)). The missing loops in the crystal structures, were built using the well-established libraries of backbone fragments (Zhang and Skolnick, 2005) and constraint space de novo reconstruction of the backbone segments (Peitsch, 1995). The catalytic domain of human TMPRSS2 (residues N146-D491; UniProt ID: O15393) was constructed using SWISS-MODEL (Waterhouse et al., 2018), based on the crystal structure of serine protease hepsin (PDB: 5CE1). A crystal structure of human furin (Y110-A408; P09958) was used as is (PDB: 5JMO) (Dahms et al., 2016).
Generation and assessment of SARS-CoV-2 Spike and protease complex models. To investigate priming of the S1/S2 site of SARS-CoV-2 Spike, protein-protein docking analysis was performed for TMPRSS2 or furin with SARS-CoV-2 Spike in the pre-fusion state. Using docking software ClusPro (Kozakov et al., 2017), a series of SARS-CoV-2 Spike and protease complexes were constructed in silico. SARS-CoV-2 Spike was set as receptor and protease as ligand. Residues in the proximity of the cleavage site from SARS-CoV-2 Spike (T676 to V687) were set as attractor sites of receptor, and the catalytic residues from TMPRSS2 (H296, D345 and 5441) or furin (D153, H194 and 5368) were set as attractor sites for ligand. For each complex, 30 clusters of conformations were obtained, upon clustering −800 models generated by ClusPro. The clusters were rank-ordered by cluster size (Kozakov et al., 2017) as recommended, and representative members from top-ranking clusters were further examined and refined Mainly, protein-protein binding free energies were calculated using PRODIGY (Xue et al., 2016); and mutagenesis and sculpting wizards in PyMOL 2.3.0 (Open Source version) (DeLano, 2002) were used to interactively refine rotamers and interactions, respectively.
Monoclonal antibodies binding to SARS-CoV-2 Spike. SEB-associated monoclonal antibodies 14G8, 6D3 and 20B1 were taken from the crystal structures of SEB bound to two neutralizing Abs, 14G8 and 6D3 (PDB: 4RGN), and one neutralizing Ab, 20B1 (PDB: 4RGM). SARS-CoV-2 S-associated neutralizing Abs were taken from the crystal structures listed in Table 4. Ab-binding poses were predicted using protein-protein docking module in ClusPro (Kozakov et al., 2017) where SARS-CoV-2 spike was set as the receptor and 6D3 as the ligand. Computations repeated with the antibody mode of ClusPro confirmed the S1/S2 cleavage site to be most favorable binding site for mAb 6D3. All docking simulations were performed using ClusPro default parameters.
aPDB IDs of the cryo-EM structures containing the indicated Ab are given in parentheses.
bEpitope residues of SARS-CoV-2 within 4 Å distance of the antibody based on the first PDB ID listed in column 3.
Model refinement and binding affinity calculations. Selective protease-Spike and mAb-Spike complexes were further refined using the refinement protocol implemented in the webserver HADDOCK 2.4 (Van Zundert et al., 2016). Refinement was performed by MD energy minimization following the protocol and default parameters provided by the webserver. Binding free energies were evaluated using the inter-residue contact-based method accessible in the webserver PRODIGY (Xue et al., 2016). The standard deviations of binding free energy were estimated based on multiple binding poses taken from docking simulations and model refinement.
Sequence alignment. Multiple sequence alignment of the variable heavy chain domain of anti-SEB Abs (6D3, 14G8 and 20B1) and anti-SARS-CoV-2 S Abs were generated by Clustal Omega (Sievers et al., 2011).
Quantification and statistical analysis. For viral inhibition assays: Quantification of immunofluorescence images was performed manually, blinded to the conditions. Five images per well were quantified and the average calculated. n=3 technical replicates (wells) per condition. Data is presented as mean+/−standard error of the mean and is representative of three independent experiments. Data were analyzed by t test (6D3 vs. isotype control) with multiple testing correction (Benjamini, Krieger and Yekutieli FDR test) using GraphPad Prism software. No methods were used to test the assumptions of the statistical approach. Statistical analysis details are found in the methods description, results and figure captions.
This application claims the benefit of U.S. Provisional Application No. 63/051,481, filed Jul. 14, 2020, which is expressly incorporated herein by reference.
This invention was made with government support under grant numbers GM103712 and AI072726 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20140234325 | Fries et al. | Apr 2014 | A1 |
| 20160039914 | McIntyre et al. | Feb 2016 | A1 |
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| Number | Date | Country | |
|---|---|---|---|
| 20220235119 A1 | Jul 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| 63051481 | Jul 2020 | US |
