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WO2022036337A1 - Compositions et méthodes pour un polypeptide recombinant imitant la protéine nucléocapside (np) du sars-cov-2 - Google Patents

Compositions et méthodes pour un polypeptide recombinant imitant la protéine nucléocapside (np) du sars-cov-2 Download PDF

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Publication number
WO2022036337A1
WO2022036337A1 PCT/US2021/054877 US2021054877W WO2022036337A1 WO 2022036337 A1 WO2022036337 A1 WO 2022036337A1 US 2021054877 W US2021054877 W US 2021054877W WO 2022036337 A1 WO2022036337 A1 WO 2022036337A1
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Prior art keywords
sars
cov
protein
amino acid
seq
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PCT/US2021/054877
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English (en)
Inventor
Serdyuk Vladimir GRIGORIEVISH
Gorlov Andrei YURIEVICH
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Diaclone Llc
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Publication of WO2022036337A1 publication Critical patent/WO2022036337A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This disclosure relates to compositions and methods for (SARS) -related coronavirus using same.
  • SARS-CoV-2 a novel strain of severe acute respiratory syndrome (SARS)-associated coronavirus named SARS-CoV-2 that causes COVID-19 spread throughout the world.
  • Coronavirus is a virus containing single-stranded RNA, of positive polarity, of approximately 30 kilobases which replicates in the cytoplasm of the host cells; the 5' end of the genome has a capped structure and the 3' end contains a polyA tail.
  • This virus is enveloped and comprises, at its surface, structures called spicules.
  • the S protein is a membrane glycoprotein (200-220 kDa) which exists in the form of spicules or spikes emerging from the surface of the viral envelope. It is responsible for the attachment of the virus to the receptors of the host cell and for inducing the fusion of the viral envelope with the cell membrane.
  • the small envelope protein (E), also called sM (small membrane), which is a nonglycosylated transmembrane protein of about 10 kDa, is the protein present in the smallest quantity in the virion. It plays an important role in the coronavirus budding process which occurs at the level of the intermediate compartment in the endoplasmic reticulum and the Golgi apparatus.
  • M protein or matrix protein (25-30 kDa) is a more abundant membrane glycoprotein which is integrated into the viral particle by an M/E interaction, whereas the incorporation of S into the particles is directed by an S/M interaction. It appears to be important for the viral maturation of coronaviruses and for the determination of the site where the viral particles are assembled.
  • the N protein or nucleocapsid protein (NP) (45-50 kDa) which is the most conserved among the coronavirus structural proteins is necessary for encapsidating the genomic RNA and then for directing its incorporation into the virion. This protein is also believed to be involved in the replication of the RNA.
  • the proteins of the viral membrane S, E and M proteins
  • S, E and M proteins are inserted into the intermediate compartment, whereas the replicated RNA (+strand) is assembled with the N (nucleocapsid) protein.
  • This protein-RNA complex then combines with the M protein contained in the membranes of the endoplasmic reticulum and the viral particles form when the nucleocapsid complex buds into the endoplasmic reticulum.
  • the virus migrates across the Golgi complex and eventually leaves the cell, for example by exocytosis.
  • the site of attachment of the virus to the host cell is understood to be at the level of the S protein.
  • Coronaviruses are believed to cause upwards of 30% of colds in humans and respiratory and digestive infections in animals, and in 2020, COVID- 19, caused by SARS CoV-2, was declared a pandemic.
  • the invention in one aspect, relates to compositions, agents, reagents, preparations, kits, uses, and methods for the detection, diagnosis, prevention, and/or treatment of SARS-related coronavirus disorders or diseases, for example, SARS CoV-2 infection.
  • the invention relates to methods for producing a polypeptide mimicking the SARS-CoV-2 nucleocapsid protein (NP).
  • the methods may include construction of plasmid DNA of recombinant polypeptide using genetic engineering technology, which according to further aspects, may contain major immunodominant epitopes of the native SARS-CoV-2 nucleocapsid protein based on the pDNP-NP-SARS-CoV2 plasmid expression vector.
  • the invention relates to a polypeptide containing epitopes peculiar to the native nucleocapsid protein (“NP-SARS-CoV-2”) for use in various compositions, agents, reagents, preparations, kits, uses, and methods, for example, those involving diagnostics, treatments and/or vaccines.
  • NP-SARS-CoV-2 native nucleocapsid protein
  • the invention relates to a method of diagnosing a SARS-related coronavirus disease or disorder in a cell of a subject, the method comprising the step of providing the cell an effective amount of at least one NP-SARS-CoV-2 protein, thereby diagnosing a SARS-related coronavirus disease or disorder.
  • the invention in another aspect, relates to a method of detecting a coronavirus in a cell of a subject, the method comprising the step of contacting the cell with an effective amount of at least one NP- SARS-CoV-2 protein, thereby detecting the coronavirus.
  • the invention in another aspect, relates to a method for the detection of a SARS-associated coronavirus in a biological sample comprising providing a NP-SARS-CoV-2 protein; providing a biological sample from a patient infected with a SARS-CoV coronavirus; contacting said protein with said biological sample; and visualizing the antigen-antibody complexes formed.
  • the method comprises an ELISA.
  • the NP-SARS-CoV-2 protein comprises or consists of the sequence of SEQ ID NO:1.
  • the invention relates to a kit for the detection of a SARS-CoV coronavirus in a biological sample comprising a NP-SARS-CoV-2 protein.
  • Also disclosed are methods for preventing viral activity in a subject comprising the step of providing to the subject an effective amount of at least one vaccine comprising an NP-SARS- CoV-2 protein, thereby preventing the viral activity.
  • Also disclosed are methods method for the treatment of a subject comprising the steps of: diagnosing the subject as having a SARS-related coronavirus disorder or disease; and administering to the subject an effective amount of at least one NP-SARS-CoV-2 protein.
  • vaccine preparations and pharmaceutical compositions comprising an effective amount of at least one NP-SARS-CoV-2 protein; and a pharmaceutically acceptable carrier.
  • kits comprising an effective amount of at least one disclosed composition, agent, preparation, or reagent, and instructions for treating or diagnosing a SARS-related coronavirus disorder or disease; and c) instructions for administering or using the least one disclosed composition, preparation, agent, or reagent in connection with the SARS-related coronavirus disorder or disease.
  • FIG. 1 is an electron micrograph of purified DIA-RP showing particles forming protein aggregates.
  • FIGS. 2A-2D are graphs showing results of immunization of rabbits with DIA-RP.
  • FIG. 3 is Immune -electron microscopy of DIA-RP showing formation of immune -complexes.
  • FIG. 4 is a graph showing the effect of monoclonal antibodies against DIA-RP on a virus replicating in tissue culture.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • references in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a weight percent (wt. %) of a component is based on the total weight of the formulation or composition in which the component is included.
  • the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the subject is a mammal.
  • a patient refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • the subject has been diagnosed with a need for treatment of one or more disorders prior to the administering step. In some aspects of the disclosed methods, the subject has been diagnosed with a need for treatment of one or more SARS-related coronavirus disorder or disease prior to the administering step. In some aspects of the disclosed method, the subject has been diagnosed with a coronavirus disease prior to the administering step. In some aspects of the disclosed method, the subject been diagnosed with a risk of SARS-COV-2 infection prior to the administering step.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease.
  • the subject is a mammal such as a primate, and, in a further aspect, the subject is a human.
  • subject also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • domesticated animals e.g., cats, dogs, etc.
  • livestock e.g., cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.
  • preventing refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. For example, preventing SARS-related coronavirus disease or disorder means reducing the incidences, delaying or reversing conditions, diseases or disorders that are related to or associated with coronavirus.
  • the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, agents, or methods disclosed herein.
  • the subject has been diagnosed with a need for detection, prevention or treatment of a SARS- related coronavirus disorder or disease prior to the administering step.
  • the phrase “identified to be in need of treatment for a disorder,” or the like refers to selection of a subject based upon need for prevention or treatment of the disorder or related condition. It is contemplated that the identification can, in one aspect, be performed by a person different from the person making the diagnosis. It is also contemplated, in a further aspect, that the administration can be performed by one who subsequently performed the administration.
  • the term “providing” refers to any method of administering or contacting a disclosed agent, preparation, compound or composition to a cell, target receptor, or other biological entity. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophy lactically; that is, administered for prevention of a disease or condition.
  • administering refers to any method of providing an agent, compound, composition, or pharmaceutical preparation to a subject.
  • Such methods include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration.
  • Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophy lac tically; that is, administered for prevention of a disease or condition.
  • contacting refers to bringing a disclosed compound and a cell, target receptor, or other biological entity together in such a manner that the compound can affect the activity of the target (e.g., receptor, cell, etc.), either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.
  • the target e.g., receptor, cell, etc.
  • the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition.
  • a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration.
  • compositions can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • a preparation can be administered in a “prophy tactically effective amount”; that is, an amount effective for prevention of a disease or condition.
  • SARS-related coronavirus disorder refers to conditions, disorders or diseases which is caused by coronavirus or is a major risk factor, for example, and without limitation, a coronavirus infection, acute respiratory syndrome, SARS-CoV-2 infection, SARS-CoV infection, COVID-19, immune-mediated disorder, or inflammatory disorder.
  • the present invention encompasses, but is not limited to the foregoing diseases or disorders.
  • infection is intended to refer to any injurious or potentially injurious condition to a cell or mammal that can be caused by, for example, a viral organism.
  • exemplary organisms include, but are not limited to coronavirus, SARS-CoV-2 infection, SARS-CoV, MERS, and the like.
  • the methods and compositions of the present disclosure can be utilized on a mammal afflicted with one or more infections, or as a preventative treatment to a mammal not afflicted with any one or more infections.
  • ECso is intended to refer to the concentration of a substance (e.g., a compound or a protein) that is required for 50% agonism or activation of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc.
  • a substance e.g., a compound or a protein
  • an ECso can refer to the concentration of a substance that is required for 50% agonism or activation in vivo, as further defined elsewhere herein.
  • ECso refers to the concentration of agonist or activator that provokes a response halfway between the baseline and maximum response.
  • IC50 is intended to refer to the concentration of a substance (e.g., a compound or a protein) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc.
  • a substance e.g., a compound or a protein
  • an IC50 can refer to the concentration of a substance that is required for 50% inhibition in vivo, as further defined elsewhere herein.
  • IC50 refers to the half maximal (50%) inhibitory concentration (IC) of a substance.
  • pharmaceutically acceptable describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.
  • the term “derivative” refers to a compound having a structure derived from the structure of a parent compound or composition (e.g., a compound, protein, or polypeptide disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds or composition, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.
  • polymorphic forms or modifications It is known that chemical substances form solids which are present in different states of order which are termed polymorphic forms or modifications.
  • the different modifications of a polymorphic substance can differ greatly in their physical properties.
  • the compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.
  • Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art.
  • the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art.
  • compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.
  • the present disclosure provides compositions, agents, kits, uses, and methods for the detection, diagnosis, and/or treatment of SARS CoV-2 and SARS-related coronavirus disorders or diseases.
  • the present disclosure provides a polypeptide containing major immunodominant epitopes peculiar to the native nucleocapsid protein (“NP-SARS-CoV-2”) for use in various compositions and methods involving diagnostics test systems, treatment and vaccines.
  • NP-SARS-CoV-2 is believed to be an early highly immunogenic antigen of SARS-CoV-2.
  • NP-SARS-CoV-2 can be used as an antigenic component in order to design diagnostic test systems, produce vaccines, as well as to carry out research and development.
  • embodiments of purified recombinant NP-SARS-CoV-2 prepared according to the present invention were advantageously found to form spherical particles about 24 nm in diameter. Accordingly, it is believed the presence of such particles is advantageous for vaccine preparations for many viral infections, for example, as virus like particles (VLPs).
  • VLPs virus like particles
  • NP-SARS-CoV-2 detection and diagnostic capability serologic examinations based on enzyme-linked immunosorbent assay (ELISA) tests and rapid tests (RTs) were initially created and tested. The created serological examinations were validated in several centers and reference laboratory specializing in test validation studies, showing complete agreement with results of commercial SARS CoV- 2 diagnostics (i.e., Abbott and Roche): 100% (CI 96%- 100%).
  • ELISA enzyme-linked immunosorbent assay
  • RTs rapid tests
  • serologic examinations surprisingly found that specific antibodies to the NP-SARS-CoV-2 antigen appear in high titers early in the course of COVID- 19 and persist for a long time; indicating the diagnostic value of this protein in developing diagnostic tests and vaccines, as well as in potential effective treatments of COVID-19.
  • the NP-SARS-COV-2 protein of SARS-CoV-2 was produced recombinantly in E. coli strain BL21 (DE3). DNA encoding the NP-SARS-COV-2 was codon optimized for expression in bacteria, the DNA was transformed into E. coli, and the protein produced was then purified.
  • NP-SARS-CoV-2 An ELISA using NP-SARS-CoV-2 was able to detect anti-SARS CoV-2 antibodies in the serum of all of patients with confirmed COVID-19 infections, suggesting all or substantially all natural infections have antibodies to NP-SARS-CoV-2, which, in turn, may indicate the importance of such antibodies as protective against coronavirus, such as SARS-CoV-2 infection. Furthermore, there is potential increased sensitivity and selectivity with the NP-SARS-COV-2 protein.
  • mice and rabbits were immunized to evaluate the ability of NP-SARS-CoV-2 to produce antibodies.
  • groups of mice had good antibody responses to DIA-RP (titer >1:64,000) as did several groups of rabbits.
  • the present invention provides a highly sensitive and specific test for the determination of the SARS-CoV-2 antigen in patients infected with the SARS-CoV-2 virus and their contacts.
  • the disclosed tests correspond to PCR test results in terms of comparable detectability with reference COVID-19 markers, but several times less expensive and it is easier and more reliable to use.
  • the present invention provides the following proteins, polypeptides, antibodies, compositions, agents, reagents, preparations and methods and kits comprising the disclosed proteins, polypeptides, antibodies, compositions, agents, reagents, preparations.
  • the invention provides methods for producing a purified modified recombinant polypeptide mimicking the SARS-CoV-2 nucleocapsid protein (NP).
  • the methods may include construction of plasmid DNA of the recombinant polypeptide using genetic engineering technology, for example, that contain major immunodominant epitopes of the native SARS- CoV-2 nucleocapsid protein based on the pDNP-NP-SARS-CoV2 plasmid expression vector.
  • the methods may comprise the step of culturing bacteria, for example in LB essential medium containing kanamycin or the like until a desire optical density is reached, for example from about 0.6-0.9 absorbance units at 600 nm.
  • the method may comprise adding isopropyl- beta-thiogalactopyranoside (IPTG) and culturing.
  • the methods may further comprises harvesting the cells, for example, by centrifuging and suspended in buffer.
  • the methods may comprise treating with lysozyme and sonicating.
  • the method may further comprise purifying and/or isolating the target polypeptide or protein, for example, by chromatography or the like.
  • the end product of the methods may be a recombinant polypeptide.
  • recombinant polypeptide may contain the sequence of the major immunodominant epitopes of the native SARS-CoV-2 nucleocapsid protein.
  • the recombinant polypeptide is NP-SARS-CoV-2.
  • the recombinant polypeptide may comprise 61 kDa molecular weight, 9.67 isoelectric point, containing about 562 amino-acid residues.
  • the recombinant polypeptide may have the general formula as follows: NH2 -(His)6 -TRX-NP-(His)6 -COOH.
  • the recombinant polypeptide may have the sequence of SEQ ID: No.1.
  • the recombinant polypeptide may have the amino acid as follows:
  • the vector employed in the methods may be constructed using various genetic engineering technology.
  • the plasmin vector may be a pDNP-NP-SARS- CoV2 bacterial plasmid vector.
  • the vector may have 6895 bps and contains a kanamycin resistance marker.
  • the vector after transformation of Escherichia coli BL21(DE3) cells and induction, can be effective to provide synthesis of a protein that is not released into the medium but is accumulated in cytoplasm of bacterial cells.
  • production of the NP-SARS-COV-2 proteins can be achieved by any technique known to the skilled artisan.
  • a method for producing SARS-CoV-2 N protein comprises the following steps: culturing a bacterium transformed with a recombinant plasmid encoding for a SARS-CoV-2 N protein of the present invention in a suitable bacterial growth medium, for example, a medium supplemented with kanamycin, such as with 50 microgram/mL kanamycin; inducing the production of SARS-CoV-2 N protein; and recovering and purifying the SARS-CoV-2 N protein.
  • the medium may further comprise chloramphenicol, for example, about 30 microgram/mL chloramphenicol.
  • the medium may be lysogeny broth (LB).
  • the bacterium is E. coli.
  • the step of inducing production of SARS-CoV-2 N protein may be performed by adding isopropyl (3-d- 1 -thiogalactopyranoside (IPTG).
  • the step of recovering the SARS-CoV-2 N protein may be performed by pelleting and breakage of bacteria (bacteria cells).
  • the step of recovering the SARS-CoV-2 N protein may be performed by pelleting and breakage of bacteria, and further recovering the soluble fraction of broken bacteria.
  • purifying SARS-CoV-2 N protein is performed by metal chelate affinity chromatography, and/or gel filtration.
  • the SARS-CoV-2 N protein made by the disclosed method disclosed may be soluble.
  • the invention provides an NP-SARS-COV-2 protein made by the processes disclosed herein, particularly in Example 1.
  • NP-SARS-COV-2 proteins and the peptides derived from these proteins and antibodies generated against them can be used for the detection and diagnosis of a SARS-related coronavirus diseases and disorders, for example, an infection (serological diagnosis (detection of specific antibodies)) or virological diagnosis (detection of viral nucleocapsid (N) protein), in particular by an immunoassay, such as an immunoenzymatic method (e.g., ELISA).
  • an infection serological diagnosis
  • virological diagnosis detection of viral nucleocapsid (N) protein
  • an immunoassay such as an immunoenzymatic method (e.g., ELISA).
  • the present invention includes methods for identifying a patient infected with a SARS-associated coronavirus or SARS-related coronavirus diseases and disorders.
  • the methods may include one or more of the following steps: providing a serum sample from the patient; contacting the serum with a NP-SARS-COV-2 protein, and visualizing the antigen-antibody complexes.
  • the antigen-antibody complexes are visualized by EIA, ELISA, RIA, or by immunofluorescence.
  • the SARS-related coronavirus diseases and disorders such as a SARS-associated coronavirus infection, is identified as SARS CoV-2 infection.
  • the patient has been shown to be infected by SARS-CoV or SARS CoV-2 by a nucleic acid detection test, such as a PCR or other nucleic acid amplification test.
  • a nucleic acid detection test such as a PCR or other nucleic acid amplification test.
  • the patient has been diagnosed or identified as being infected with a SARS-related coronavirus disease and disorder without detection of the virus by PCR or another nucleic acid amplification technique.
  • the present invention provides a composition comprising an NP-SARS- COV-2 protein or method for use of an NP-SARS-COV-2 protein for detection of antibodies against a SARS coronavirus and/or diagnosis of a SARS coronavirus infection, in a biological sample.
  • the SARS coronavirus is a SARS CoV-2 coronavirus.
  • the ability of NP-SARS-COV- 2 to bind with high affinity to anti-SARS CoV-2 antibodies can allows its use in such methods, such as for diagnostics of a SARS CoV-2 infection.
  • antibodies and antibody fragments according to the invention are useful for the direct detection and diagnosis of SARS-related coronavirus diseases and disorders, such as SARS-associated coronavirus infection, and for the detection of a SARS-CoV in a biological sample.
  • detection of the nucleocapsid (N) protein of a SARS coronavirus may be carried out by suitable techniques, for example EIA, ELISA, RIA, immunofluorescence, in a biological sample collected from a likely infected subject.
  • the subject has been shown to be infected by SARS-CoV or SARS CoV-2 by a nucleic acid detection test, such as a PCR or other nucleic acid amplification test.
  • the invention provides a method for detection of SARS-related coronavirus diseases and disorders, such from a SARS-associated coronavirus, from a biological sample.
  • the method may comprise the steps of: contacting a biological sample from a patient infected with a SARS-CoV or SARS-CoV-2 coronavirus with an anti-NP-SARS-COV-2 antibody, and visualizing the antigen-antibody complexes formed.
  • the antigen-antibody complexes may be visualized by EIA, ELISA, RIA, or by immunofluorescence, or the like.
  • the NP-SARS-COV-2 protein may be attached to an appropriate support, for example, and without limitation, a microplate or a bead.
  • the invention provides a method comprising the steps of: contacting a biological sample from a subject, preferably a human, infected with a SARS-CoV or SARS CoV-2 coronavirus with an NP-SARS-COV-2 protein, which is attached to an appropriate support, for example a microplate or bead, under conditions effective to allow binding to occur; washing the support to remove unbound antibodies; adding a detection reagent that binds to the immunoglobulins bound to NP- SARS-COV-2 protein; and detecting the NP-SARS-COV-2 protein-antibody complexes formed.
  • the invention provides a method for the detection of antibodies directed to a SARS-associated coronavirus in a biological sample, the method comprising the steps of: providing a NP-SARS-COV-2 protein: providing a biological sample from a patient infected with a SARS- CoV coronavirus; contacting said NP-SARS-COV-2 protein with said biological sample; and visualizing the antigen-antibody complexes formed.
  • the method comprises an ELISA.
  • the NP-SARS-COV-2 protein comprises or consists of the sequence of SEQ ID NO:1.
  • the patient is infected with a SARS-CoV-2 coronavirus.
  • the proteinantibody complexes may be detected with an antibody or an antibody fragment that binds to human immunoglobulins.
  • the detection reagent may comprise a label selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label.
  • the detection reagent is a labeled antibody or antibody fragment that binds to human immunoglobulins.
  • labels may include a fluorescent label, such as FITC, a chromophore label, an affinity-ligand label, an enzyme label, such as alkaline phosphatase, horseradish peroxidase, luciferase or (3 galactosidase, an enzyme cofactor label, a hapten conjugate label, such as digoxigenin or dinitrophenyl, a Raman signal generating label, a magnetic label, a spin label, an epitope label, such as the FLAG or HA epitope, a luminescent label, a heavy atom label, a nanoparticle label, an electrochemical label, a light scattering label, a spherical shell label, semiconductor nanocrystal label, wherein the label can allow visualization with or without a secondary detection molecule.
  • a fluorescent label such as FITC
  • a chromophore label such as alkaline phosphatase, horseradish peroxidase, luciferase or (3
  • labels may include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin/biotin, avidin/biotin or an antigen/antibody complex including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexe
  • suitable enzymes such as horse
  • the antibody or an antibody fragment that binds to human immunoglobulins binds specifically to IgG, IgA, and IgM. In some embodiments, the antibody or an antibody fragment that binds to human immunoglobulins binds specifically to IgG, IgA, or IgM.
  • the term “antibodies” is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof, such as F(ab')2 and Fab fragments, single-chain variable fragments (scFvs), single-domain antibody fragments (VHHs or Nanobodies), bivalent antibody fragments (diabodies), as well as any recombinantly and synthetically produced binding partners.
  • the disclosed methods may comprise comparing the results obtained with a patient serum to positive and negative controls.
  • Positive controls can include: Serum from animals (e.g., rabbit, mouse, etc.) immunized with NP-SARS-COV-2 protein as described herein, and NP-SARS- COV-2 protein as described herein.
  • the disclosed methods can comprise the use of NP-SARS-COV-2 proteins to detect novel coronaviruses that do not cross-react with seasonal (non-pathogenic) coronaviruses.
  • the NP-SARS-COV-2 protein comprises or consists of the amino acid sequence of SEQ ID NO: 1.
  • the method may comprise an immunocapture method.
  • the method may comprise attaching a first monoclonal or polyclonal antibody or a fragment thereof, directed against the NP-SARS-COV-2 (capture antibody), incubating the antibody with a biological sample containing a NP-SARS-COV-2, and detecting the antigen-antibody complexes formed, preferably with a monoclonal antibody (visualizing antibody).
  • the biological sample is mixed with the visualizing monoclonal antibody prior to its being brought into contact with the capture antibody.
  • the antibody used for the capture of the NP-SARS-COV-2 protein is a monoclonal antibody produced a hybridoma.
  • a visualizing molecule may be a radioactive atom, a dye, a fluorescent molecule, a fluorophore, an enzyme: a visualizing particle may be for example: colloidal gold, a magnetic particle or a latex bead.
  • the present invention also relates to a method for the detection of a SARS- associated coronavirus infection, from a biological sample, by indirect IgG ELISA using the NP-SARS- COV-2 protein.
  • method may comprise plates that are sensitized with an NP-SARS- COV-2 protein solution at a concentration of between 0.5 and 4 pg/ml, preferably to 2 pg/ml, in a 10 mM PBS buffer pH 7.2, phenol red at 0.25 ml/1.
  • microtiter plates are coated by incubation overnight at 4° C. with 5 pg/ml of N proteins.
  • the invention provides methods for preventing, reducing the occurrence of, or reducing the severity of a disease, comprising: administering a vaccine as described herein, to a subject; wherein the administration prevents, reduces the occurrence of, or reduces the severity of the disease.
  • the disease comprises an infection.
  • the disease comprises a bacterial, fungal, or viral infection.
  • the viral infection comprises an influenza infection.
  • the viral infection is a coronavirus infection.
  • the viral infection is coronavirus disease 2019 (CO VID 19).
  • the subject is a mammal or human subject.
  • the administration comprises administration by one or more needles or microneedles. In some embodiments, the administration comprises administration by a preformed liquid syringe. In some embodiments, the administration comprises intranasal, intradermal, intramuscular, skin patch, topical, oral, subcutaneous, intraperitoneal, intravenous, or intrathecal administration.
  • the administration comprises administering a dose of 1 pg, 10 pg, 25 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 50 ng, 100 ng, 250 ng, 500 ng, 1 pg, 10 pg, 50 pg, 100 pg, 500 pg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1 g of the seVLP or vaccine, or a range of doses defined by any two of the aforementioned doses.
  • the invention provides methods for determining an effectiveness of a vaccine, comprising: obtaining a sample obtained from a subject who has been administered a vaccine, the sample comprising a presence or an amount of a virus; providing a substrate comprising an ACE2 or fragment thereof capable of binding to a virus protein; contacting the substrate with the sample to bind virus or protein virus in the sample to the ACE2 or fragment thereof; detecting virus or protein virus bound to the ACE2 or fragment thereof of the substrate; and determining the presence or amount of the virus in the sample based on the detected virus or protein virus bound to the ACE2 or fragment thereof of the substrate, thereby determining the effectiveness of the vaccine.
  • the sample is from a subject.
  • the sample comprises blood, serum, or plasma.
  • the virus is a coronavirus.
  • the virus is a SARS-CoV-2.
  • the virus protein is a NP-SARS-CoV-2 protein.
  • the amount of virus in the sample is decreased compared to another sample obtained from the subject before the subject was administered the vaccine.
  • the amount of virus in the sample is increased compared to another sample obtained from the subject before the subject was administered the vaccine.
  • Some embodiments further comprise recommending or providing a virus treatment to the subject based on the amount of the virus in the sample or the effectiveness of the vaccine.
  • the virus treatment comprises a coronavirus treatment such as a CO VID- 19 treatment.
  • the vaccine comprises a VLP.
  • the invention provides methods for determining an effectiveness of a vaccine, comprising: obtaining a sample obtained from a subject who has been administered a vaccine, the sample comprising a presence or an amount of anti-virus antibodies; providing a substrate comprising a virus protein or fragment thereof capable of binding to the anti- virus antibodies; contacting the substrate with the sample to bind anti-virus antibodies in the sample to the virus protein or fragment thereof; detecting anti-virus antibodies bound to the virus protein or fragment thereof of the substrate; and determining the presence or amount of the anti- virus antibodies in the sample based on the detected antivirus antibodies bound to the virus protein or fragment thereof of the substrate, thereby determining the effectiveness of the vaccine.
  • the sample is from a subject.
  • the sample comprises blood, serum, or plasma.
  • the virus is a coronavirus.
  • the virus is a SARS-CoV-2.
  • the virus protein is a NP-SARS-CoV-2 protein.
  • the amount of anti-virus antibodies in the sample is decreased compared to another sample obtained from the subject before the subject was administered the vaccine.
  • the amount of anti-virus antibodies in the sample is increased compared to another sample obtained from the subject before the subject was administered the vaccine.
  • Some embodiments further comprise recommending or providing a virus treatment to the subject based on the amount of the anti-virus antibodies in the sample or the effectiveness of the vaccine.
  • the virus treatment comprises a coronavirus treatment such as a COVID-19 treatment.
  • the vaccine comprises a VLP.
  • the invention relates to a recombinant vector for expression of sell N protein of SARS-CoV-2.
  • the recombinant vector can be a vector for eukaryotic or prokaryotic expression, such as a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector.
  • an expression vector as defined herein is chosen to enable the production of a N protein or poly epitope, either in vitro or in vivo.
  • the recombinant vector for expression of NP-SARS-CoV-2 may be pET-24a (+) as contained in the bacteria strain E.
  • the recombinant vector for expression of NP-SARS-CoV-2 is pET-19b (+) as contained in the bacteria strain E.coli BL21(DE3)- GOLD.
  • the expression vector encodes a protease cleavage site, such as Xa cleavage site, inserted between N protein coding sequence and a protein purification Tag, such as GST tag.
  • a protease cleavage site is positioned to remove the tag, for example, after purification.
  • the expression vector can comprise transcription regulation regions (including promoter, enhancer, ribosome binding site (RBS), polyA signal), a termination signal, a prokaryotic or eukaryotic origin of replication and/or a selection gene.
  • the features of the promoter can be easily determined by the man skilled in the art in view of the expression needed, i.e., constitutive, transitory or inducible (e.g., IPTG), strong or weak, tissue-specific and/or developmental stage-specific promoter.
  • the vector can also comprise sequence enabling conditional expression, such as sequences of the Cre/Lox system or analogue systems.
  • the expression vector is a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector, or any expression vector.
  • An expression vector as defined herein is chosen to enable the production of a protein or polyepitope, either in vitro or in vivo.
  • the nucleic acid molecules according to the invention can be obtained by conventional methods, known per se, following standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc., Library of Congress, USA). For example, they may be obtained by amplification of a nucleic sequence by PCR or RT-PCR or alternatively by total or partial chemical synthesis.
  • the vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods which are known per se.
  • Numerous vectors into which a nucleic acid molecule of interest may be inserted in order to introduce it and to maintain it in a host cell are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of the sequence in extrachromosomal form or alternatively integration into the chromosomal material of the host), and on the nature of the host cell.
  • the NP-SARS-CoV-2 protein comprises the amino acid sequence of the SARS CoV-2 nucleocapsid protein.
  • the NP-SARS-COV-2 protein may comprise one or more of: a sequence of a major immunodominant epitopes of the disclosed native SARS-CoV-2 nucleocapsid protein, for example, with 61 kDa molecular weight, 9.67 isoelectric point, containing 562 amino-acid residues and has the general formula as follows: NH 2 -(His)s -TRX-NP-(His)s -COOH.
  • the NP-SARS-COV-2 protein comprises the following amino acid sequence: (SEQ ID NO: 1) 1 MGSSHHHHHH SSGLVPRGSH MSDKIIHLTD DSFDTDVLKA DGAILVDFWA
  • the NP-SARS-COV-2 protein is produced by bacteria transformed with the expression vector pET32a(+) or pGEX-4Tl or with the expression vector pEX-C-His.
  • the expression vector is contained within bacterial strain Escherichia coli B BL21 (DE3). In further aspects, the expression vector is contained within one of the following bacterial strains Escherichia coli BL21 (DE3) pLysS or Escherichia coli BL21-GOLD or Escherichia coli B BL21 (DE3) pDIA17.
  • the present invention provides “isolated or purified” NP-SARS-COV-2 proteins.
  • isolated or purified mean modified “by the hand of humans” from the natural state. To this end, if an object exists in nature, it is said to be isolated or purified if it is modified or extracted from its natural environment or both. For example, a polynucleotide or a protein/peptide naturally present in a living organism is neither isolated nor purified: on the other hand, the same polynucleotide or protein/peptide separated from coexisting molecules in its natural environment, obtained by cloning, amplification and/or chemical synthesis is isolated for the purposes of the present invention.
  • a polynucleotide or a protein/peptide which is introduced into an organism by transformation, genetic manipulation or by any other method is “isolated” even if it is present in said organism.
  • the term purified as used in the present invention means that the proteins/peptides according to the invention are essentially free of association with the other proteins or polypeptides, as is for example the product purified from the culture of recombinant host cells or the product purified from a non-recombinant source.
  • Various techniques can be used to obtain purified protein according to the invention, such as, for example metal chelate binding chromatography and gel filtration.
  • the present invention encompasses an immune complex formed of a polyclonal or monoclonal antibody or antibody fragment as defined herein, and of a NP-SARS-COV-2 protein.
  • the invention also encompasses various antibodies against NP-SARS- COV-2.
  • antibodies may comprise polyclonal or monoclonal antibodies generated in rabbits or mice.
  • antibodies may comprise a polyclonal or monoclonal antibody or fragment thereof directed against NP-SARS-COV-2 protein.
  • antibodies can be obtained by immunization of an animal with a NP-SARS-COV-2 protein.
  • the antibodies can serve as reagents to bind native NP-SARS-COV-2 proteins of patients in immunoassays.
  • the antibodies can serve as positive control reagents to bind isolated and purified NP-SARS-COV-2 proteins in immunoassays of patients.
  • the antibodies can be used to determine and adjust the concentration of NP-SARS-CoV-2 bound to ELISA plates for serum dilution.
  • the invention encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and fragments thereof (e.g., Fab, Fv, scFv) directed against the N protein, for example, a NP-SARS-CoV-2 protein disclosed herein.
  • polyclonal antibodies monoclonal antibodies, chimeric antibodies, and fragments thereof (e.g., Fab, Fv, scFv) directed against the N protein, for example, a NP-SARS-CoV-2 protein disclosed herein.
  • the expression chimeric antibody is understood to mean, in relation to an antibody of a particular animal species or of a particular class of antibody, an antibody comprising all or part of a heavy chain and/or of a light chain of an antibody of another animal species or of another class of antibody.
  • purified proteins may be used to produce antibodies by conventional techniques.
  • recombinant or synthetic proteins or peptides may be used to produce antibodies by conventional techniques.
  • antibodies can be synthetic, semi-synthetic, monoclonal, or polyclonal and can be made by techniques well known in the art. Such antibodies specifically bind to proteins and polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding). Purified or synthetic proteins and peptides can be employed as immunogens in producing antibodies immunoreactive therewith. The proteins and peptides contain antigenic determinants or epitopes that elicit the formation of antibodies.
  • these antigenic determinants or epitopes can be either linear or conformational (discontinuous).
  • Linear epitopes may be composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes may be composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding. Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes can be less than the number of available epitopes.
  • epitopes can be identified by any of the methods known in the art. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology. In some embodiments,
  • antibodies are defined to be specifically binding if they bind proteins or polypeptides with a Ka of greater than or equal to about 107 M-l. Affinities of binding partners or antibodies can be readily determined using conventional techniques known in the art.
  • polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, alpaca, camels, rabbits, mice, or rats, using procedures that are well known in the art.
  • a purified protein or polypeptide that is appropriately conjugated is administered to the host animal typically through parenteral injection.
  • the immunogenicity can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant, to simulate analogous vaccine preparations corresponding to the drugs administered to humans.
  • an adjuvant for example, Freund's complete or incomplete adjuvant
  • Examples of various assays useful for such determination include procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme- linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays.
  • CIEP countercurrent immuno-electrophoresis
  • ELISA enzyme- linked immunosorbent assays
  • sandwich assays examples include procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme- linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays.
  • monoclonal antibodies can be readily prepared using procedures known in the art.
  • host animals such as mice
  • Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given an intravenous boost of the protein or polypeptide.
  • ABSC antibody capture
  • mice are later sacrificed, and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell.
  • the fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig.
  • a label such as a labeled protein or polypeptide
  • a labeled protein or polypeptide is added to each well followed by incubation. Positive wells can be subsequently detected. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column.
  • monoclonal antibodies of the present invention can be produced using alternative techniques.
  • binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody.
  • antigen-binding fragments of such antibodies which can be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments.
  • Antibody fragments and derivatives produced by genetic engineering techniques are also provided.
  • monoclonal antibodies of the present invention may include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies.
  • humanized antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans.
  • a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • antibodies produced by genetic engineering methods such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, can be used.
  • Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art.
  • sdAb single-domain antibodies
  • NANOBODIES single-domain antibodies
  • a sdAb is a fragment consisting of a single monomeric variable antibody domain that can bind selectively to a specific antigen.
  • sdAbs may be from heavychain antibodies found in camelids (VHH fragments), or cartilaginous fishes (VNAR fragments), or are obtained by splitting dimeric variable domains into monomers.
  • the invention provides various vaccine and vaccine preparations, such as a virus like particle (VLP), comprising a disclosed coronovirus protein, or a fragment thereof.
  • a “vaccine” refers to or otherwise comprises a preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of disease, such as an infectious disease.
  • the immunogenic material is a VLP disclosed herein.
  • vaccines elicit both prophylactic (preventative) and therapeutic responses.
  • methods of administration vary according to the vaccine, or include inoculation, ingestion, intranasal, intradermal, or other forms of administration.
  • vaccines are administered with an adjuvant to enhance the immune response.
  • a VLP refers to or comprises an enveloped structure resembling a virus made up of one of more viral structural proteins, but which lacks a viral genome.
  • VLPs lack a viral genome and are non-infectious.
  • VLPs are divided into nonenveloped and eVLPs.
  • enveloped VLPs include a lipid membrane.
  • the VLP presents a properly folded, functional antigen.
  • the VLPs present HA that binds to receptors on epithelial cells or red blood cells.
  • the VLPs present NA and have enzymatic activity that cleaves sialic acids.
  • the VLPs comprise synthetic enveloped VLPs (seVLPs).
  • the coronavirus protein comprises a spike (S) protein, an envelope (E) protein, a membrane protein (M), or a nucleocapsid (N) protein.
  • the coronavirus protein is NP-SARS-CoV-2.
  • the coronavirus protein comprises an amino acid sequence that is 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 99.9%, 100%, or a range of percentages defined by any two of the aforementioned percentages, identical to SEQ ID NO: 1, or a fragment thereof.
  • the coronavirus protein comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, or a range defined by any of the aforementioned integers, amino acid substitutions, deletions, and/or insertions, compared to any of SEQ ID NO: 1, or a fragment thereof.
  • a vaccine preparation may comprise a vaccine like particle comprising (a) a synthetic lipid bilayer comprising a first lipid and a second lipid; (b) an anchor molecule embedded in the lipid bilayer; and (c) a disclosed SARS-CoV-2 protein bound to the anchor molecule.
  • the anchor molecule may comprise a transmembrane protein, a lipid- anchored protein, or a fragment or domain thereof.
  • the anchor molecule comprises a hydrophobic moiety.
  • the anchor molecule comprises a prenylated protein, fatty acylated protein, a glycosylphosphatidy linositol-linked protein, or a fragment thereof.
  • the VLP further comprises a synthetic lipid vesicle comprising the lipid bilayer.
  • the lipid bilayer comprises an inner surface and an outer surface.
  • the antigen is presented on the outer surface of the lipid vesicle.
  • the antigen is presented on the inner surface of the lipid vesicle.
  • the VLP is a seVLP and the lipid bilayer is in the form of a synthetic lipid vesicle.
  • the VLP is in the form of a synthetic membrane virus-like particle (smVLP) comprising a nanodisc.
  • the nanodisc has a diameter of between 5-200 nM.
  • the nanodisc comprises an amphiphilic polymethacrylate (PMA) copolymer.
  • the nanodisc comprises styrene- maleic acid lipid particles (SMALPs).
  • the nanodisc comprises a diisobutylenemaleic acid (DIBMA) co-polymer.
  • the PMA copolymer is toroidal.
  • the SMALPs are toroidal.
  • the DIBMA co- polymer is toroidal.
  • the nanodisc comprises an amphiphilic toroidal polymethacrylate (PMA) copolymer, SMALP, or DIBMA co-polymer.
  • a vaccine may comprise: a VLP as described herein, and a pharmaceutically acceptable excipient, carrier, and/or adjuvant.
  • the excipient comprises an anti-adherent, a binder, a coating, a color or dye, a disintegrant, a flavor, a glidant, a lubricant, a preservative, a sorbent, a sweetener, or a vehicle.
  • the vaccine comprises the adjuvant.
  • the adjuvant comprises a Toll-like receptor (TLR) agonist such as imiquimod, Flt3 ligand, monophosphoryl lipid A (MLA), or an immuno stimulatory oligonucleotide such as a CpG oligonucleotide.
  • TLR Toll-like receptor
  • MVA monophosphoryl lipid A
  • the adjuvant comprises imiquimod.
  • the vaccine is formulated in a solvent or liquid such as a saline solution, a dry powder, or as a sugar glass.
  • the vaccine is lyophilized.
  • the vaccine is formulated for intranasal, intradermal, intramuscular, topical, oral, subcutaneous, intraperitoneal, intravenous, or intrathecal administration.
  • the vaccine comprises a dose of 1 pg, 10 pg, 25 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 50 ng, 100 ng, 250 ng, 500 ng, 1 pg, 10 pg, 50 pg, 100 pg, 500 pg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1 g of the seVLP, or a range of doses defined by any two of the aforementioned doses.
  • the vaccine comprises a dose of 25 pL, 50 pL, 100 pL, 250 pL, 500 pL, 750 pL, 1 nL, 5 nL, 10 nL, 15 nL, 20 nL 25 nL, 50 nL, 100 nL, 250 nL, 500 nL, 1 pL, 10 pL, 50 pL, 100 pL, 500 pL, 1 mL, or 5 mL of the vaccine, or a range of doses defined by any two of the aforementioned doses.
  • VLPs comprising: (a) a synthetic lipid bilayer comprising a first lipid and a second lipid; (b) an anchor molecule embedded in the lipid bilayer; and (c) a NPSARS-CoV-2 protein bound to the anchor molecule.
  • the SARS-CoV-2 protein is bound directly to the anchor molecule, or wherein the SARS-CoV-2 protein comprises the anchor molecule.
  • the SARS-CoV-2 protein comprises a N protein.
  • the N protein comprises an amino acid sequence that is greater than 85% or 95% identical to SEQ ID NO: 1.
  • the spike protein comprises an amino acid sequence that has no more than 10 amino acid substitutions, deletions, or insertions, compared to SEQ ID NO: 1.
  • vaccines comprising the VLP, and a pharmaceutically acceptable excipient, carrier, or adjuvant.
  • the adjuvant comprises imiquimod.
  • the vaccine is formulated for injection by a microneedle.
  • the vaccine is lyophilized.
  • the vaccine is formulated as a sugar glass.
  • vaccination methods comprising administering the vaccine to a subject in need thereof.
  • synthetic enveloped virus-like particles comprising: (a) a synthetic lipid vesicle comprising a lipid bilayer having an inner surface and an outer surface; (b) an anchor molecule embedded in the lipid bilayer; and (c) a disclosed NP-SARS-CoV- 2 protein bound to the anchor molecule.
  • the NP-SARS-CoV-2 protein is presented on the outer surface of the lipid vesicle. In some embodiments, the NP-SARS- CoV-2 protein is presented on the inner surface of the lipid vesicle.
  • smVLPs comprising: (a) a synthetic nanodisc comprising a lipid bilayer comprising an inner surface and an outer surface; (b) an anchor molecule embedded in the lipid bilayer; and (c) a disclosed NP-SARS-CoV-2 protein bound to the anchor molecule.
  • the nanodisc comprises a 5-200 nM diameter.
  • the nanodisc comprises an amphiphilic toroidal polymethacrylate (PMA) copolymer, SMALP, DIBMA co-polymer, or non-immunogenic mimetic peptides of an alpha helix of ApoA.
  • PMA amphiphilic toroidal polymethacrylate
  • the SARS-CoV-2 protein comprises an SI or S2 spike protein.
  • the smVLP is formulated as a sugar glass for injection. Also disclosed herein, in certain embodiments, are devices loaded with a disclosed vaccine as described herein.
  • virus-like particle VLPs comprising: a synthetic lipid bilayer comprising a first lipid and a second lipid; an anchor molecule embedded in the lipid bilayer; and a SARS-CoV-2 protein bound to the anchor molecule.
  • the first lipid comprises a phosphatidylcholine species.
  • the first lipid comprises DOPC.
  • the second lipid comprises phosphatidylethanolamine species.
  • the second lipid comprises DOPE.
  • the lipid bilayer comprises the first lipid and the second lipid at a predetermined ratio between 1:0.25 and 1:4.
  • the lipid bilayer further comprises cholesterol or DC-cholesterol, or a derivative thereof. In some embodiments, the lipid bilayer comprises the cholesterol or DC-cholesterol, or a derivative thereof at a ratio of 0-30 mol% in relation to the first lipid or the second lipid.
  • a vaccine comprising the VLP, and a pharmaceutically acceptable excipient, carrier, or adjuvant. In some embodiments, the adjuvant comprises imiquimod. In some embodiments, the vaccine is formulated for injection by a needle or microneedle. In some embodiments, the vaccine is lyophilized. Some embodiments comprise a vaccination method comprising administering the vaccine to a subject in need thereof.
  • seVLPs comprising: a synthetic lipid vesicle comprising a lipid bilayer having an inner surface and an outer surface; an anchor molecule embedded in the lipid bilayer; and a NP-SARS-CoV-2 protein bound to the anchor molecule.
  • the NP-SARS-CoV-2 protein is presented on the outer surface of the lipid vesicle. In some embodiments, the NP-SARS-CoV-2 protein is presented on the inner surface of the lipid vesicle.
  • kits can be prepared from the disclosed compounds, products, and pharmaceutical compositions. It is also understood that the disclosed kits can be employed in connection with the disclosed methods of using.
  • the present invention encompasses at least the following aspects:
  • a method for producing a SARS-CoV-2 nucleocapsid (N) protein comprising the following steps: culturing a bacterium transformed with a recombinant plasmid encoding for a SARS-CoV- 2 N protein comprising the sequence of SEQ ID NO:1; inducing the production of the SARS-CoV-2 N protein; recovering and purifying NP-SARS-CoV-2 protein.
  • Aspect 2 The method of aspect 1, wherein recovering of SARS-CoV-2 N protein is performed by pelleting and breakage of bacteria.
  • Aspect 3 The method of aspects 1-2, wherein purifying of SARS-CoV-2 N protein is performed by metal chelate affinity chromatography, and/or gel filtration.
  • Aspect 4 The method of aspects 1-3, wherein the SARS-CoV-2 N protein comprises an amino acid sequence that is 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 99.9%, 100%, or a range of percentages defined by any two of the aforementioned percentages, identical to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 5 The method of aspects 1-4, wherein the SARS-CoV-2 N protein comprises an amino acid sequence that is 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 99.9%, 100%, or a range of percentages defined by any two of the aforementioned percentages, identical to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 6 The method of aspects 1- 5, wherein the the SARS-CoV-2 N protein comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, or a range defined by any of the aforementioned integers, amino acid substitutions, deletions, and/or insertions, compared to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 7 The method of aspects 1-6, wherein the the SARS-CoV-2 N protein consist of an amino acid sequence of SEQ ID NO: 1.
  • Aspect 9 The SARS-CoV-2 N protein of aspect 8, wherein said SARS-CoV-2 N protein comprises a 61 kDa molecular weight, a 9.67 isoelectric point, and has the general formula: NH2 -(His)6 -TRX-NP- (His)6 -COOH.
  • a method for the detection of a SARS-related coronavirus infection in a biological sample comprising: providing a SARS-CoV-2 nucleocapsid (N) protein expressed and purified from E. coli strain BL21 (DE3) transformed with a recombinant plasmid; providing a biological sample from an individual or a patient suspected to be infected with a severe acute respiratory syndrome-associated coronavirus (SARS-CoV) coronavirus; contacting said NP-SARS-CoV-2 protein with said biological sample; and visualizing the antigen-antibody complexes formed.
  • Aspect 11 The method of aspect 10, further comprising detection of at least one of: IgG, IgM or IgA.
  • Aspect 12 The method of aspect 10, further comprising detection of IgG, IgM and IgA.
  • Aspect 13 The method of aspect 10, comprising an ELISA, lateral flow immunoassays, bead based immunoassays, or multiplex bead based immunoassays.
  • Aspect 14 The method of aspect 10, wherein the NP-SARS-CoV-2 protein comprises the sequence of SEQ ID NO:1.
  • Aspect 15 The method of aspect 10, wherein the SARS-CoV-2 N protein consists of the sequence of SEQ ID NO:1.
  • Aspect 16 The method of aspect 10, wherein the SARS-CoV-2 N protein comprises an amino acid sequence that is 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 99.9%, 100%, or a range of percentages defined by any two of the aforementioned percentages, identical to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 17 The method of aspect 10, wherein the SARS-CoV-2 N protein comprises an amino acid sequence that is 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 99.9%, 100%, or a range of percentages defined by any two of the aforementioned percentages, identical to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 18 The method of aspect 10, wherein the SARS-CoV-2 N protein comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, or a range defined by any of the aforementioned integers, amino acid substitutions, deletions, and/or insertions, compared to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 19 The method of aspect 10, wherein the SARS-CoV-2 N protein comprises the protein of aspect 8.
  • Aspect 20 A recombinant SARS-CoV-2 N protein, or a fragment thereof, comprising an amino acid sequence of SEQ ID NO: 1.
  • Aspect 21 The protein of aspect 20, wherein said protein comprises an amino acid sequence that is 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 99.9%, 100%, or a range of percentages defined by any two of the aforementioned percentages, identical to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 22 The protein of aspect 20, wherein said protein comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, or a range defined by any of the aforementioned integers, amino acid substitutions, deletions, and/or insertions, compared to any of SEQ ID NO: 1, or a fragment thereof.
  • a vaccine comprising the SARS-CoV-2 N protein any preceding aspects, and a pharmaceutically acceptable excipient, carrier, and/or adjuvant.
  • Aspect 24 The vaccine of aspect 23, wherein the excipient comprises an antiadherent, a binder, a coating, a color or dye, a disintegrant, a flavor, a glidant, a lubricant, a preservative, a sorbent, a sweetener, or a vehicle.
  • Aspect 25 The vaccine of aspect 23, wherein the adjuvant comprises a Toll-like receptor (TLR) agonist such as imiquimod, Flt3 ligand, monophosphoryl lipid A (MLA), or an immuno stimulatory oligonucleotide such as a CpG oligonucleotide.
  • TLR Toll-like receptor
  • MLA monophosphoryl lipid A
  • an immuno stimulatory oligonucleotide such as a CpG oligonucleotide.
  • Aspect 26 The vaccine of aspect 23, wherein the vaccine is formulated for intranasal, intradermal, intramuscular, topical, oral, subcutaneous, intraperitoneal, intravenous, or intrathecal administration.
  • Aspect 27 The vaccine of aspect 23, wherein the vaccine comprises a dose of 1 pg, 10 pg, 25 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 50 ng, 100 ng, 250 ng, 500 ng, 1 mg, 10 mg, 50 mg, 100 mg, 500 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1 g of the seVLP, or a range of doses defined by any two of the aforementioned doses.
  • Aspect 28 The vaccine of aspect 23, wherein the vaccine comprises a dose of 25 pL, 50 pL, 100 pL, 250 pL, 500 pL, 750 pL, 1 nL, 5 nL, 10 nL, 15 nL, 20 nL 25 nL, 50 nL, 100 nL, 250 nL, 500 nL, 1 mL, 10 mL, 50 mL, 100 mL, 500 mL, 1 mL, or 5 mL of the vaccine, or a range of doses defined by any two of the aforementioned doses.
  • Aspect 29 The vaccine of aspect 23, wherein the SARS-CoV-2 N protein comprises the protein of aspect 8.
  • Aspect 30 A kit for the detection of a SARS-CoV coronavirus infection in a biological sample comprising a NP-SARS-CoV-2 protein expressed and purified from E. coli BL21 (DE3) transformed with a recombinant plasmid.
  • Aspect 31 The kit of aspect 30, comprising serum from an animal immunized with a NP-SARS-CoV-2 protein.
  • Aspect 32 The kit of aspect 30, wherein the SARS-CoV-2 N protein comprises an amino acid sequence that is 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 99.9%, 100%, or a range of percentages defined by any two of the aforementioned percentages, identical to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 33 The kit of aspect 30, wherein the SARS-CoV-2 N protein comprises an amino acid sequence that is 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 99.9%, 100%, or a range of percentages defined by any two of the aforementioned percentages, identical to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 34 The kit of aspect 30, wherein the SARS-CoV-2 N protein comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, or a range defined by any of the aforementioned integers, amino acid substitutions, deletions, and/or insertions, compared to any of SEQ ID NO: 1, or a fragment thereof.
  • Aspect 35 The kit of aspect 30, wherein the SARS-CoV-2 N protein consist of an amino acid sequence of SEQ ID NO: 1.
  • Aspect 36 The kit of aspect 30, wherein the SARS-CoV-2 N protein comprises the protein of aspect 8.
  • a purified modified recombinant polypeptide mimicking the SARS-CoV-2 nucleocapsid protein (NP) was produced in Example 1.
  • the nucleotide sequence of the immunogenic antigen is synthesized and cloned in the expression vector pET-24a (+) between EcoRI and Hind III restriction sites, which encodes the SARS-CoV-2 nucleocapsid protein that contains the sequence of the major immunodominant epitopes of the native NP SARS-CoV-2 nucleocapsid protein and is controlled by the induced expression system. Cloning may be confirmed by sequencing. Constructs are codon-optimized for expression in E. coli. Optimization process includes codon adaptation, mRNA de novo synthesis and stability, transcription and translation efficiency.
  • ligase mixture in Escherichia coli was transformed; transformants were selected in LB plates containing kanamycin; the required clone was been identified, called as pDNP-NP-SARS- CoV2, and transformed into Escherichia coli BL21(DE3).
  • kanamycin 50 pg/ml
  • Isopropyl-beta-thiogalactopyranoside IPTG is added to the cells to achieve concentration of 0.33 mM and culturing is continued for another 3 hours. Thereafter the cells are harvested by centrifuging at 8000 rpm for 10 min at 4°C and suspended in TE buffer (0.01 Tris-HCl, 0.001 M EDTA, pH 8.0), then the cells are treated with lysozyme and sonicated; the lysate is centrifuged at 12000 rpm for 30 min. The resulting precipitate of inclusion bodies is washed with the same buffer by centrifuging 5 times. The washed inclusion bodies are dissolved in 20 mM Tris-HCl buffer, pH 8, 8 M urea and the recombinant protein is purified by metal chelate column chromatography with Ni-IDA agarose.
  • the resulting end product was the target recombinant polypeptide, containing the sequence of the major immunodominant epitopes of the native SARS-CoV-2 nucleocapsid protein.
  • the protein is detected as a major band by polyacrylamide gel electrophoresis and Western blotting with anti-SARS- CoV-2 serum, its molecular weight is 61 kDa, isoelectric point is 9.67. It contains 562 amino-acid residues and has the general formula as follows: NH2-(His)6- TRX-NP-(His)6-COOH.
  • the resulting polypeptide has the sequence:
  • the pDNP-NP-SARS-CoV2 bacterial plasmid vector was constructed using genetic engineering technology, and has 6895 bps and contains a kanamycin resistance marker. After transformation of Escherichia coli BL21(DE3) cells and induction of IPTG, it provides synthesis of a protein that is not released into the medium but is accumulated in cytoplasm of bacterial cells.
  • the isolated recombinant protein was tested by enzyme-linked immunosorbent assay using serum of people who have had antibodies to the SARS-CoV-2 structural protein. As shown in FIG. 1, embodiments of purified recombinant NP-SARS-CoV-2 prepared according to the present invention were advantageously found to form spherical particles about 24 nm in diameter.
  • Example 2 various immunization and challenge studies were performed. Initially, NP- SARS-CoV-2 was evaluated to study the ability of NP-SARS-CoV-2 to produce antibodies in laboratory animals (mice and rabbits). First, mice were immunized with NP-SARS-CoV-2 recombinant Nucleoprotein (N) prepared according to Example 1 at days 0 (prime), and/or 28 (boost). Naive (buffer only) mice and/or adjuvant-treated mice are used as controls. Serum sample are collected from mice and tested. Mice were found to have good antibody responses to NP-SARS-CoV-2 (titer >1:64,000).
  • N Nucleoprotein
  • FIG. 3 shows an immuno-electron microscopy photograph of the NP-SARS-CoV-2 protein surrounded by authentic antibodies. The data indicate that the obtained antibodies correspond to particles (corpuscles) of the NP-SARS-CoV-2 protein. Accordingly, this immunization study of mice indicates ability to obtain hybridomas and to produce monoclonal antibodies to the NP-SARS-CoV-2 protein.
  • Figure 4 shows the effect of monoclonal antibodies against NP-SARS-CoV-2 on the SARS-CoV-2 virus replicating in tissue culture. As the data show, the virus was neutralized in the culture of infected human cells by mouse monoclonal antibodies to NP-SARS-CoV-2.
  • the black line shows "positive control," which is the effect of the virus on cells without an inhibitory agent.
  • the percentage of neutralization reached 80% in the case of antibodies M 001- 20 and about 60% in the other two antibody samples.
  • Three samples of monoclonal antibodies to the NP-SARS- CoV-2 antigen showed virus neutralization at a concentration of 200 pg/ml and at a more significant dilution of 50 pg/ml. The data provides significant evidence that the antibodies produced against the NP- SARS-CoV-2 protein can prevent viral replication.
  • NP-SARS-CoV-2 protein sequence was found along with SARS-CoV-2, SARS-CoV, and MERS and found the NP-SARS-CoV-2 protein to have highly conservative fragments in common with the three agents studied (SARS-CoV-2, SARS-CoV, and MERS). Several fragments coincided with known immunodominant regions. This observation suggests that NP-SARS-CoV-2-based vaccine in accordance with the present invention may induce antibodies that may have cross-reactivity between SARS-CoV-2, SARS-CoV-1, and MERS coronaviruses.

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Abstract

L'invention concerne des compositions, agents, réactifs, préparations, kits, utilisations et méthodes pour la détection, le diagnostic, la prévention et/ou le traitement de troubles ou de maladies à coronavirus associés au SARS utilisant des protéines de nucléocapside du SARS-CoV-2 et des anticorps se liant à ces protéines. Le présent abrégé est proposé à titre d'outil d'exploration à des fins de recherche dans cette technique particulière et n'est pas destiné à limiter la présente invention.
PCT/US2021/054877 2020-08-13 2021-10-13 Compositions et méthodes pour un polypeptide recombinant imitant la protéine nucléocapside (np) du sars-cov-2 WO2022036337A1 (fr)

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CN117534750A (zh) * 2023-10-16 2024-02-09 遵义医科大学珠海校区 一种抗新型冠状病毒核衣壳蛋白的抗体或其抗原结合片段及其应用
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