WO2021207303A1

WO2021207303A1 - Immune stimulation against coronavirus infections

Info

Publication number: WO2021207303A1
Application number: PCT/US2021/026103
Authority: WO
Inventors: Claude Nicolau; Reynald THINARD; Ruth Greferath; Melanie CHEVALIER
Original assignee: Alsatech, Inc.
Priority date: 2020-04-07
Filing date: 2021-04-07
Publication date: 2021-10-14

Abstract

The present invention is related to methods and compositions for the therapeutic and diagnostic use in the treatment of infections that are caused by or associated with coronaviruses (e.g., SARS-CoV-2). In particular, the present invention provides novel methods and compositions for eliciting a highly specific and highly effective immune response in a patient, which is capable of preventing or alleviating coronavirus infections, or symptoms associated with coronavirus infections, including the novel SARS-CoV-2 infection, COVID-19.

Description

IMMUNE STIMULATION AGAINST CORONAVIRUS INFECTIONS

FIELD

The present invention is related to methods and compositions for the therapeutic and diagnostic use in the prevention and/or mitigation of infections which are caused by or associated with coronaviruses, including the novel severe acute respiratory syndrome (SARS)-CoV-2 (also known as 2019-nCoV).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Application No. 63/006,401, filed April 7, 2020, the contents of which are included by reference in their entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (Filename: "ALS-011PC_ST25.txt”; Date recorded: April 7, 2021; File size: 18,821 bytes).

BACKGROUND

Coronaviruses (CoVs) are a large family of enveloped, positive-sense, single-stranded RNA viruses that infect a wide range of vertebrates. They are extensively found in bats but can also be found in many other birds and mammals, including humans. CoVs can cause a variety of diseases such as enteritis in pigs and cows and upper respiratory disease in chickens. In humans, CoVs tend to cause mild to moderate upper respiratory tract infections, such as the common cold.

Over the past several decades there have been outbreaks of severe, and sometimes fatal, respiratory illnesses that were later found to be caused by novel, human pathogenic CoVs. For example, unique coronaviruses have been determined to be responsible for serious infectious outbreaks of potentially fatally atypical pneumonia. In December 2019, one such coronavirus-related infection was defined as coronavirus disease 19 (COVID-19) and was found to be caused by novel severe acute respiratory syndrome (SARS)-CoV-2. This CoV is similar to the CoV that was responsible for the SARS pandemic that occurred in 2002. Angiotensin Converting Enzyme 2 (ACE2) is the host cell receptor responsible for mediating infection by SARS-CoV-2.

These novel, human pathogenic coronavirus strains exhibit strong virulence and are quickly passed from human to human. While infection with these CoVs typically produces mild symptoms, for certain individuals responses are more severe, with death occurring in extreme cases due to gradual respiratory failure as the result of alveolar damage.

Currently, there is no approved treatment for COVID-19, and vaccines are in their infancy. Accordingly, there is an urgent need for SARS-CoV-2 vaccines that could prevent and/or mitigate COVID-19 and related infections. SUMMARY OF THE INVENTION

The present invention provides novel methods and compositions for eliciting a highly specific and highly effective immune response in an organism, but particularly within a patient or subject, which is capable of preventing and/or mitigating coronavirus-related infections (e.g., COVID-19 or severe acute respiratory syndrome (SARS)), or the symptoms associated with coronavirus-related infections.

In particular, the present invention provides novel methods and compositions for preventing a mammal from contracting infection by coronavirus, such as infection by SARS-CoV-2 which is responsible for COVID-19, comprising administering a composition that elicits anti-SARS-CoV-2 antibodies that disrupt the interaction between virus and the Angiotensin Converting Enzyme 2 (ACE2) receptor. In one aspect, the present invention contemplates compositions and methods for the prevention or mitigation of a coronavirus-related infection comprising administering to a patient at risk of suffering from such an infection or a patient suffering from such an infection an antigenic composition comprising one or more copies of a palmitoylated SARS-CoV-2 peptide antigen reconstituted in a liposome. In various embodiments, the SARS-CoV-2 peptide antigen is (a) pre-formed by on-resin standard automated peptide synthesis and (b) then modified by on-resin grafting of a palmitoyl moiety to the terminal amino acid residues of the pre-formed SARS-CoV-2 peptide. In further embodiments, the SARS-CoV-2 peptide is a fragment of the SARS-CoV-2 receptor binding domain (RBD), or the spike protein, optionally wherein the SARS-CoV-2 peptide is a fragment of the SARS- CoV-2 RBD comprising amino acid residues 485-502 of the SARS-CoV-2 surface glycoprotein (SEQ ID NO: 2). In embodiments, the SARS-CoV-2 peptide is a fragment of a variant SARS-CoV-2 RBD, or the spike protein,

In various embodiments, the present invention contemplates use of compositions and methods for prevention or mitigation of a coronavirus-related infection via administration of the antigenic composition described herein. In some embodiments, administration of the antigenic composition results in disruption or reduction in the interaction between the ACE2 receptor and the coronavirus; disruption or reduction in SARS-CoV-2 receptor recognition and/or SARS- CoV-2 membrane fusion with a host cell; or disruption or reduction in SARS-CoV-2 infection of a host cell.

In another aspect, the present invention contemplates an antigenic construct comprising one or more copies of a palmitoylated SARS-CoV-2 peptide antigen reconstituted in a liposome. In various embodiments, the SARS-CoV-2 peptide antigen is pre-formed by on-resin standard automated peptide synthesis and modified by on-resin grafting of a palmitoyl moiety to the terminal amino acid residues of the pre-formed SARS-CoV-2 peptide antigen. In some embodiments, the SARS-CoV-2 peptide antigen comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions. In further embodiments, the peptide further comprises at least one or at least two palmitoyl moieties on the N terminus or C terminus or both termini. For example, the antigenic construct of the present invention can include a palmitoylated SARS-CoV-2 peptide antigen that is H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, and wherein "Pal” is a palmitoyl moiety. For example, the antigenic construct of the present invention can include a palmitoylated SARS-CoV-2 peptide antigen that is H(Pal)Lys-(Pal)Lys- SEQ ID NO: 4-(Pal)Lys-(Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, and wherein "Pal” is a palmitoyl moiety.

Other aspects and embodiments of the invention will be apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts amino acid sequences contemplated by the present invention.

Figure 2 shows the inhibition percentage results of the SARS-CoV-2 Surrogate Virus Neutralization experiment.

Figure 3 depicts the percent inhibition relative to the positive control of the test of the "Anti- ALS 1&2 purif” group in concentrations of 10ng/microL, 20ng/ microL, 30ng/ microL, 40ng/ microL, 80ng/ microL. Percent inhibition is calculated as 1 - ABS value of Sample / ABS value of Negative Control) x 100%.

Figure 4 shows percent inhibition of Anti-ALS1, Anti-ALS2, Anti-ALSB, Pre-ALS1, Pre-ALS2, and Pre-ALSB, each diluted 1 :9 with sample buffer.

Figure 5 depicts percent inhibition of BLCO serum dilutions.

Figure 6 shows percent inhibition of the "ALS-B” group and the Non-immunized group in the concentration of 100 ng/pL.

Figure 7 depicts percent inhibition of the "Anti- ALS 1 &2 purif” group in concentrations of 10 ng/pL, 20 ng/pL, 30 ng/pL, 40 ng/pL, 50 ng/pL, and 100 ng/pL.

Figures 8A-B depict the cytopathic effects of SARS-CoV-2 exhibited in Vero E6 cell cultures when the Vero E6 cells were inoculated with oropharyngeal swab sample IAIU (10⁴ copies/pL). Figure 8A shows Vero cell cultures in negative control, and Figure 8B shows the cytopathic effects consisting of rounding, detachment of cells, blebbing and intense vacuolization, 4 days after inoculation.

DETAILED DESCRIPTION

It is an object of the invention to provide a therapeutic or preventative vaccine composition and method of producing such a composition for the prevention and/or mitigation of infections, diseases and disorders which are caused by or associated with coronaviruses (e.g., severe acute respiratory syndrome (SARS)-CoV-2). In one aspect, the present invention contemplates administering to a patient in need thereof a composition that elicits anti-SARS-CoV-2 antibodies that disrupt the interaction between coronavirus and receptor. In particular, various embodiments contemplate compositions and methods for the prevention or mitigation of a coronavirus-related infection comprising administering to a patient at risk of suffering from such an infection or a patient suffering from such an infection an antigenic composition comprising one or more copies of a palmitoylated SARS-CoV-2 peptide antigen that is attached to, or incorporated in or reconstituted in a carrier particle/adjuvant such as, for example, a liposome. Specifically, the antigenic composition comprises a SARS-CoV-2 peptide that is a fragment of the SARS-CoV-2 RBD, optionally wherein the SARS-CoV-2 peptide is a fragment of the SARS-CoV-2 RBD comprising amino acid residues 485-502 of the SARS- CoV-2 surface glycoprotein, optionally wherein the peptide is a fragment of the RBD of the SARS-CoV-2 surface glycoprotein.

In various embodiments, the full SARS-CoV-2 surface glycoprotein comprises the amino acid sequence of SEQ ID NO: 2 (Accession No. YP_009724390).

In various embodiments, the full ACE2 receptor comprises the amino acid sequence of SEQ ID NO: 1 (Accession No. NM_001371415). See, Yan et al., "Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2,” Science ol. 367(6485): 1444-1448, which is incorporated herein in its entirety.

In another aspect, the present invention contemplates compositions and methods for the prevention or mitigation of a coronavirus-related infection comprising administering to a patient at risk of suffering from such an infection or a patient suffering from such an infection an antigenic composition comprising one or more copies of a palmitoylated SARS- CoV-2 peptide antigen reconstituted in a liposome. In various embodiments, the SARS-CoV-2 peptide antigen is (a) pre-formed by on-resin standard automated peptide synthesis and (b) then modified by on-resin grafting of a palmitoyl moiety to the terminal amino acid residues of the pre-formed SARS-CoV-2 peptide. In further embodiments, the SARS- CoV-2 peptide is a fragment of the SARS-CoV-2 RBD, optionally wherein the SARS-CoV-2 peptide is a fragment of the SARS-CoV-2 RBD of the s1 subunit of the SARS-CoV-2 surface glycoprotein.

In various embodiments, the present invention contemplates use of compositions and methods for prevention or mitigation of a coronavirus-related infection via administration of the antigenic composition described herein. In some embodiments, administration of the antigenic composition results in disruption or reduction in the interaction between the ACE2 receptor and the coronavirus; disruption or reduction in SARS-CoV-2 receptor recognition and/or SARS- CoV-2 membrane fusion with a host cell; or disruption or reduction in SARS-CoV-2 infection of a host cell. In various embodiments, the antigenic SARS-CoV-2 peptide fragment comprises amino acid residues F486, N487, Q493, Q498, T500, N501 of the SARS-CoV-2 surface glycoprotein having the amino acid sequence of SEQ ID NO: 2 that interact with the a1 helix of the ACE2 receptor.

In a further embodiment, the invention provides a therapeutic vaccine composition and method of producing such a composition for the prevention and/or mitigation of infections which are caused by or associated with coronaviruses, including a group of infections associated with severe acute respiratory syndromes, using an antigenic SARS-CoV-2 peptide fragment, but particularly an SARS-CoV-2 peptide fragment comprising the amino acid sequence of SEQ ID NO: 3 OR SEQ ID NO: 4. Also contemplated by the present invention is a peptide fragment which is essentially identical to the above mentioned fragments and has substantially the same biological activity of said fragments, but particularly a peptide fragment that is a conservatively modified variant of said fragments in that the alterations result in the substitution and/or deletion of one or more amino acid residues, particularly of between one to six amino acid residues, optionally between one to 5 amino acid residues, between one to 4 amino acid residues, between one to 3 amino acid residues, or between one to 2 amino acid residues, with a chemically similar amino acid residue. Conservative substitution tables providing functionally similar amino acids are well known in the art and disclosed herein below. The conservative substitution is optionally to be made such that the overall net charge of the peptide and also the charge distribution over the peptide molecule remains essentially the same.

It is also an object of the invention to provide a method for the preventing and/or mitigation of infections, diseases and disorders which are caused by or associated with coronaviruses, a family of viruses associated with upper respiratory tract infections in humans, including, but not limited to, COVID-19 and SARS, by administering to a patient or subject, a vaccine composition according to the invention and as described herein.

The vaccine composition according to the present invention upon administration to a patient or subject, results mainly in the generation of anti-SARS-CoV-2 antibodies. In a further embodiment of the invention, the vaccine according to the present invention, upon administration to a patient or subject, leads to a disruption of the interaction between the coronavirus and the ACE2 receptor. For example, in a particular embodiment, the vaccine according to the present invention, upon administration to a patient or subject, leads to a disruption of the interaction between SARS-CoV-2 and the ACE2 receptor. In another embodiment, the vaccine according to the present invention, upon administration to a patient or subject, leads to a disruption or reduction of the interaction between SARS-CoV and the ACE2 receptor, e.g. via the generation and subsequent binding of anti-SARS-CoV-2 antibodies.

The present invention further provides a vaccine composition, which, upon administration to a patient or subject, induces the generation of an antibody in the treated patient or subject that directly and specifically binds to SARS-CoV- 2. In a specific embodiment of the invention, the antibody binds to an epitope within an epitopic region of SARS-CoV- 2, e.g. within a region that interacts with the ACE2 receptor. The antibodies which are induced by the vaccine composition according to the invention and which can be obtained from an immunized animal or a hybridoma cell line producing said antibodies, are also part of the invention. In embodiments, the anti SARS-CoV antibodies recognize the conformation of the SARS-CoV S1 protein that engages the ACE2 receptor. Accordingly, in various embodiments, the present antibodies bind to SARS-CoV-2 S1 protein, when it is in the conformation that engages the ACE2 receptor, thus reducing or ablating the interaction of the SARS-CoV-2 virus and the ACE2 receptor, and, optionally reducing or ablating SARS-CoV-2 infectivity. Supramolecular Antigenic Construct and Compositions

It is an objective of the present invention to provide a method for preventing, treating or mitigating the effects of coronavirus-related infections, including, but not limited to, COVID-19 or SARS, by administering a supramolecular antigenic construct or composition according to the present invention, but particularly a vaccine composition comprising such a supramolecular antigenic construct according to the invention, to a patient or subject affected by such a disorder and thus in need of such a treatment.

In one aspect, the present invention contemplates an antigenic construct comprising one or more copies of a palmitoylated SARS-CoV-2 peptide antigen reconstituted in a liposome. In various embodiments, the SARS-CoV-2 peptide antigen is pre-formed by on-resin standard automated peptide synthesis and modified by on-resin grafting of a palmitoyl moiety to the terminal amino acid residues of the pre-formed SARS-CoV-2 peptide antigen. In some embodiments, the SARS-CoV-2 peptide antigen comprises the amino acid sequence of SEQ ID NO: 3 OR SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions. In further embodiments, the peptide further comprises at least one or at least one, or at least two, or at least three, or at least four, or at least five palmitoyl moieties on the N terminus or C terminus or both termini. For example, the antigenic construct of the present invention can include a palmitoylated SARS-CoV-2 peptide antigen that is H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, and wherein "Pal” is a palmitoyl moiety. For example, the antigenic construct of the present invention can include a palmitoylated SARS-CoV-2 peptide antigen that is H(Pal)Lys-(Pal)Lys- SEQ ID NO: 4-(Pal)Lys- (Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, and wherein "Pal” is a palmitoyl moiety.

In another embodiment of the present invention a method is provided for the preparation of a vaccine composition for inducing an immune response in an organism, in particular a patient or subject affected by such an infection, disorder, disease or condition and thus in need of such a treatment, for preventing, treating or mitigating the effects of coronavirus-related infections, including, but not limited to, COVID-19 or SARS.

In still a further embodiment of the present invention a method is thus provided for the preparation of a therapeutic vaccine composition for preventing, treating or mitigating the effects of coronavirus-related infections, including, but not limited to, COVID-19 or SARS, comprising formulating an antibody according to the invention in a pharmaceutically acceptable form.

In a specific embodiment, the present invention makes use of an antigen presentation that results in enhanced exposure and stabilization of a preferred antigen conformation, which ultimately leads to a highly specific immune response and results in the generation of antibodies with unique properties. In an embodiment, the present invention provides immunogenic compositions comprising a supramolecular antigenic construct comprising a SARS-CoV-2 peptide antigen according to the invention and as described herein before representative of the N-terminal part of the SARS-CoV-2 peptide, which antigenic peptide is modified such that it is capable of maintaining and stabilizing a defined conformation of the antigen, particularly a conformation which is characterized by a balanced proportion of random coil, alpha-helical and beta-sheet portions. This defined conformation leads to the induction of a strong and highly specific immune response upon introduction into a patient or subject.

The term "supramolecular antigenic construct" refers to an antigenic construct according to the present invention and as described herein. In particular, "supramolecular antigenic construct" refers to an antigenic construct comprising an SARS-CoV-2 peptide antigen according to the invention and as described herein, specifically the SARS-CoV-2 peptide fragment of SEQ ID NO: 3 OR SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletion. For example, the supramolecular antigenic construct of the present invention can include a palmitoylated SARS-CoV-2 peptide antigen that is H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, and wherein "Pal” is a palmitoyl moiety. . For example, the supramolecular antigenic construct of the present invention can include a palmitoylated SARS-CoV-2 peptide antigen that is H(Pal)Lys-(Pal)Lys- SEQ ID NO: 4-(Pal)Lys-(Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, and wherein "Pal” is a palmitoyl moiety.

In various embodiments, the antigenic peptide is presented as attached to, or incorporated or reconstituted in a carrier such as, for example, a vesicle, a particulate body or molecule but, particularly, a liposome. More particularly, the antigenic peptide according to the invention is modified by a lipophilic or hydrophobic moiety, that, without wishing to be bound by theory, facilitates insertion into the lipid bilayer of the liposome carrier/immune adjuvant, particularly by a lipophilic or hydrophobic moiety including, but not limited to, a fatty acid, a triglyceride or a phospholipid, but especially a fatty acid, a triglyceride or a phospholipid, wherein the fatty acid carbon back bone has at least 10 carbon atoms which functions as an anchor for the peptide in the liposome bilayer and has a dimension that leads to the peptide being positioned and stabilized in close proximity to the liposome surface. In embodiments, the fatty acid, a triglyceride or a phospholipid has at least 10 carbon atoms, or at least 11 carbon atoms, or at least 12 carbon atoms, or at least 13 carbon atoms, or at least 14 carbon atoms, or at least 15 carbon atoms, or at least 16 carbon atoms, or at least 17 carbon atoms, or at least 18 carbon atoms, or at least 19 carbon atoms, or at least 20 carbon atoms. In embodiments, the fatty acid, a triglyceride or a phospholipid is a decanoic (capric), undecanoic, dodecanoic (lauric), tridecanoic, tetradecanoic (myristic), pentadecanoic, hexadecanoic (palmitic), heptadecanoic (margaric), octadecanoic (stearic), nonadecanoic, or eicosanoic (arachidic) moiety or a derivative thereof. The supramolecular antigenic constructs according to the present invention may be used for the preparation of a vaccine composition for inducing an immune response in an organism, in particular a mammal or human, for preventing, treating or mitigating the effects of coronavirus-related infections, including, but not limited to, COVID-19 or SARS.

It is thus an objective of the present invention to provide a method for preventing, treating or mitigating the effects of coronavirus-related infections, including, but not limited to, COVID-19 or SARS, by administering a supramolecular antigenic construct according to the present invention, but particularly a vaccine composition comprising such a supramolecular antigenic constructs according to the invention to a patient or subject, at risk of or affected by such an infection and thus in need of such prevention and/or mitigation.

In still a further embodiment of the present invention, a method is thus provided for the preparation of a composition for preventing, treating or mitigating the effects of coronavirus-related infections, including, but not limited to, COVID- 19 or SARS, comprising formulating an antibody according to the invention in a pharmaceutically acceptable form.

In a specific embodiment, the present invention makes use of an antigen presentation that results in enhanced exposure and stabilization of a preferred antigen conformation, which ultimately leads to a highly specific immune response and results in the generation of antibodies with unique properties.

In one embodiment, the present invention provides immunogenic compositions comprising a supramolecular antigenic construct comprising a SARS-CoV-2 peptide antigen according to the invention and as described herein before representative of the N-terminal part of the SARS-CoV-2 peptide, which antigenic peptide is modified such that it is capable of maintaining and stabilizing a defined conformation of the antigen, particularly a conformation which is characterized by a balanced proportion of random coil, alpha-helical and beta-sheet portions. This defined conformation leads to the induction of a strong and highly specific immune response upon introduction into a patient or subject.

The immunogenic compositions of the present invention may comprise liposomes made by reconstituting liposomes in the presence of purified or partially purified or modified antigenic peptides according to the invention. Additionally, peptide fragments may be reconstituted into liposomes. The present invention also includes antigenic peptide fragments modified so as to increase their antigenicity. For example, antigenic moieties and adjuvants may be attached to or admixed with the peptide. Examples of antigenic moieties and adjuvants include, but are not limited to, lipophilic muramyl dipeptide derivatives, nonionic block polymers, aluminum hydroxide or aluminum phosphate adjuvant, and mixtures thereof.

In various embodiments, the antigenic SARS-CoV-2 peptide is modified by a lipophilic or hydrophobic moiety, that facilitates insertion into the lipid bilayer of the liposome carrier/immune adjuvant, particularly by a lipophilic or hydrophobic moiety which functions as an anchor for the peptide in the liposome bilayer and has a dimension that leads to the peptide being positioned and stabilized in close proximity to the liposome surface. In a further embodiment of the invention, the lipophilic or hydrophobic moiety is a fatty acid, a triglyceride or a phospholipid, but especially a fatty acid, a triglyceride or a phospholipid, wherein the fatty acid carbon back bone has at least 10 carbon atoms. Particularly, the lipophilic or hydrophobic moiety is a fatty acid with a carbon backbone of at least approximately 14 carbon atoms and up to approximately 24 carbon atoms, with each individual number of carbon atom falling within this range also being part of the present invention. More particularly, the lipophilic or hydrophobic moiety has a carbon backbone of at least 14 carbon atoms, but especially 16 carbon atoms. Examples of hydrophobic moieties include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid. In a specific embodiment of the present invention the lipophilic or hydrophobic moiety is palmitic acid.

In still a further embodiment of the invention the hydrophobic moiety is palmitic acid and the liposome preparation may in addition contain an adjuvant such as, for example, lipid A, alum, calcium phosphate, interleukin 1, and/or microcapsules of polysaccharides and proteins, but particularly a detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, or alum.

In a specific embodiment of the invention, 2 or more of the palmitoylated SARS-CoV-2 peptide antigen molecules modified by covalently attached palmitoyl residues at each end of the peptide are reconstituted in a single liposome.

The present invention provides novel methods and immunogenic compositions comprising an immunogenic antigenic peptide, which, upon administration to a patient or subject suffering from a coronavirus-related infection, including, but no limited to, COVID-19 or SARS, induces an immune response in said patient or subject. The treatment with the therapeutic vaccine according to the invention leads to prevention and/or mitigation of the coronavirus-related infection.

Also part of the invention is a palmitoylated SARS-CoV-2 peptide antigen according to the invention and as described herein before, specifically a palmitoylated SARS-CoV-2 peptide fragment of the SARS-CoV-2 RBD of the SARS-CoV- 2 s1 subunit surface glycoprotein. In some embodiments, the SARS-CoV-2 peptide fragment is palmitoylated and comprises the amino acid sequence of SEQ ID NO: 3 OR SEQ ID NO: 4. In further embodiments, the SARS-CoV-2 peptide fragment is modified by covalently attaching palmitoyl residues, particularly between 1 and 2, between 1 and 4, or between 2 and 4 palmitoyl residues, coupled to each terminus of the peptide antigen via one or more, but particularly via one or two suitable amino acid residues such as lysine, glutamic acid or cysteine, or any other amino acid residue that can be suitably used for coupling a palmitoyl residue to the antigenic peptide.

Carrier proteins that can be used in the supramolecular antigenic construct compositions of the present invention include, but are not limited to, maltose binding protein "MBP"; bovine serum albumin "BSA"; keyhole lympet hemocyanin "KLH"; ovalbumin; flagellin; thyroglobulin; serum albumin of any species; gamma globulin of any species; syngeneic cells; syngeneic cells bearing la antigens; and polymers of D- and/or L-amino acids. In the supramolecular antigenic construct according to the present invention, the liposome may have a dual function in that it can be used as a carrier comprising the supramolecular construct as described herein before and, at the same time, function as an adjuvant to increase or stimulate the immune response within the target patient or subject to be treated with the therapeutic vaccine according to the invention. It is also to be understood that the supramolecular antigenic construct compositions of the present invention can further comprise additional adjuvants including, but not limited to, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and other adjuvants such as, for example, lipid A, alum, calcium phosphate, interleukin 1, and/or microcapsules of polysaccharides and proteins, but particularly a detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, or alum, further preservatives, diluents, emulsifiers, stabilizers, and other components that are known and used in vaccines of the prior art. Moreover, any adjuvant system known in the art can be used in the composition of the present invention. Such adjuvants include, but are not limited to, Freund's incomplete adjuvant, Freund's complete adjuvant, polydispersed beta-(1,4) linked acetylated mannan ("Acemannan"), TITERMAX® (polyoxyethylene-polyoxypropylene copolymer adjuvants from CytRx Corporation), modified lipid adjuvants from Chiron Corporation, saponin derivative adjuvants from Cambridge Biotech, killed Bordetella pertussis, the lipopolysaccharide (LPS) of gram-negative bacteria, large polymeric anions such as dextran sulfate, and inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate.

One way of achieving the formation and stabilization of the desired conformation of the antigenic peptide is by presenting the antigenic peptide attached to, or incorporated or reconstituted, partially or fully, into a carrier such as, for example, a vesicle, a particulate body or molecule or any other means that can suitably serve as a carrier/adjuvant for the antigenic peptide. In a specific embodiment of the invention, the antigenic peptide is attached to, or incorporated or reconstituted in the carrier through weak interactions such as, for example, van der Waal's, hydrophobic or electrostatic interaction, or a combination of two or more of said interactions, such that the peptide is presented with a specific conformation, which is maintained and stabilized by restricting said antigenic peptide in its three dimensional freedom of movement so that conformational changes are prevented or severely restricted.

When a vesicle, a particle or a particulate body is used as a carrier/adjuvant such as, for example, a liposome, the composition of the antigenic peptide may be chosen such that its overall net charge is identical to that of the carrier/adjuvant surface to which the peptide is attached. Electrostatic repulsion forces being effective between the identically charged carrier/adjuvant surface and the antigenic peptide, but particularly the identically charged carrier surface and the amino acid residues constituting the antigenic peptide and more particularly the identically charged carrier surface and the identically charged amino acid residues comprised in the antigenic peptide, may lead to the antigenic peptide taking on a defined, highly specific and stabilized conformation which guarantees a high biological activity. As a result, the antigenic peptide is exposed and presented in a conformation that is highly biologically active in that it allows the immune system of the target organism to freely interact with the antigenic determinants contained in the antigenic construct in the biologically active conformation, which leads to a strong and conformation-specific immune response, resulting in, for example, a high antibody titer in the target organism.

By carefully coordinating the overall net charges of the antigenic peptide on the one side and of the carrier to which the peptide becomes attached, incorporated or reconstituted in on the other side, the antigenic peptide is presented exposed on, or in close proximity to, the carrier surface in a conformation that is induced and stabilized by electrostatic repulsion forces being effective between the identically charged carrier surface and the antigenic peptide, but particularly the identically charged carrier surface and the amino acid residues constituting the antigenic peptide and more particularly the identically charged carrier surface and the identically charged amino acid residues comprised in the antigenic peptide. This results in a presentation of the antigenic construct such that is freely accessible to the immune defense machinery of the target organism and thus capable of inducing a strong and highly specific immunogenic response upon administration to a patient or subject. The immunogenic response may be further increased by using a liposome as a carrier, which liposome may function as an adjuvant to increase or stimulate the immune response within the target patient or subject to be treated with the therapeutic vaccine according to the invention. Optionally, the liposome may, in addition, contain a further adjuvant such as, for example, lipid A, alum, calcium phosphate, interleukin 1, and/or microcapsules of polysaccharides and proteins, but particularly a detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, or alum.

In a specific embodiment of the invention an antigenic peptide according to the invention and described herein before, particularly an antigenic peptide the overall net charge of which is negative, is used reconstituted in a liposome, particularly a liposome the constituents of which are chosen such that the net overall charge of the liposome head group is negative. In particular, the liposome is composed of constituents selected from the group consisting of dimyristoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl ethanolamine (DMPEA), dimyristoyl phosphatidyl glycerol (DMPG) and cholesterol and, optionally, further contains monophosphoryl lipid A or any other adjuvant that can be suitably used within the scope of the present invention such as, for example, alum, calcium phosphate, interleulin 1, and/or microcapsules of polysaccharides and proteins.

In another specific embodiment of the invention a modified peptide antigen according to the invention and as described herein before is provided covalently bound to an anchor-type molecule which is capable of inserting into the carrier/adjuvant thereby fixing the peptide to the carrier/adjuvant and presenting it on or in close proximity to the surface of a carrier/adjuvant molecule such that electrostatic forces can become effective as described herein before.

When liposomes are used as a carrier/adjuvant, the antigenic peptide construct generally has a hydrophobic tail that inserts into the liposome membrane as it is formed. Additionally, antigenic peptides can be modified to contain a hydrophobic tail so that it can be inserted into the liposome. The supramolecular antigenic constructs of the present invention generally comprise peptides modified to enhance antigenic effect wherein such peptides may be modified via pegylation (using polyethylene glycol or modified polyethylene glycol), or modified via other methods such by palmitic acid as described herein before, poly-amino acids (e.g. poly-glycine, poly-histidine), poly-saccharides (e.g. polygalacturonic acid, polylactic acid, polyglycolide, chitin, chitosan), synthetic polymers (polyamides, polyurethanes, polyesters) or co-polymers (e.g.. poly(methacrylic acid) and N-(2-hydroxy) propyl methacrylamide) and the like.

In a specific embodiment of the invention, antigenic peptides according to the invention and as described herein before are provided, which are modified to contain a hydrophobic tail so that said peptides can be inserted into the liposome. In particular, the SARS-CoV-2 peptide may be modified by a lipophilic or hydrophobic moiety that facilitates insertion into the lipid bilayer of the carrier/adjuvant. The lipophilic or hydrophobic moieties of the present invention may be fatty acids, triglycerides and phospholipids, particularly fatty acids, triglycerides and phospholipids, wherein the fatty acid carbon back bone has at least 10 carbon atoms particularly lipophilic moieties having fatty acids with a carbon backbone of at least approximately 14 carbon atoms and up to approximately 24 carbon atoms, more particularly hydrophobic moieties having a carbon backbone of at least 14 carbon atoms. Examples of hydrophobic moieties include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, linolenic acid and cholesterol or DSPE. In a specific embodiment of the invention the hydrophobic moiety is palmitic acid.

Palmitoylation, while providing an anchor for the peptide in the liposome bilayer, due to the relative reduced length of the Ci_6:o fatty acid moiety leads to the peptide being presented exposed on or in close proximity to the liposome surface. Therefore, the cells processing the antigen will have to take up the entire liposome with the peptide.

In another embodiment of the invention, PEG is used in the preparation of a supramolecular construct, wherein the free PEG terminus is covalently attached to a molecule of phosphatidylethanolamine (where the fatty acid can be: myristic, palmitic, stearic, oleic etc. or combination thereof). This supramolecular structure may be reconstituted in liposomes consisting of phospholipids and cholesterol (phosphatidylethanol amine, phosphatidyl glycerol, cholesterol in varied molar ratios. Other phospholipids can be used. Lipid A is used at a concentration of approximately 40 pg/pmole of phospholipids.

Yet another object of the present invention is to provide vaccine compositions comprising supramolecular antigenic constructs comprising an antigenic SARS-CoV-2 peptide according to the invention and as described herein, which peptide is modified so as to enhance the antigenic effect of the SARS-CoV-2 peptide. In various embodiments, the antigenic SARS-CoV-2 peptide comprises the amino acid sequence of SEQ ID NO: 3 OR SEQ ID NO: 4. In various embodiments, the SARS-CoV-2 peptide is modified via pegylation (using polyethylene glycol or modified polyethylene glycol), or modified via other methods such by poly-amino acids (e.g. poly-glycine, poly-histidine), poly-saccharides (e.g. polygalacturonic acid, polylactic acid, polyglycolide, chitin, chitosan), synthetic polymers (polyamides, polyurethanes, polyesters) or co-polymers (poly(methacrylic acid) and N-(2-hydroxy) propyl methacrylamide) and the like.

In another embodiment of the invention, the SARS-CoV-2 peptide antigen according to the invention is a palmitoylated SARS-CoV-2 peptide fragment. In various embodiments, the SARS-CoV-2 peptide antigen is modified by covalently attaching palmitoyl residues at each end of the peptide to result in between 1 and 4 residues reconstituted in a liposome. This antigenic palmitoylated construct can be used for the treatment of a coronavirus-related infection, including, but not limited to, COVID-19 or SARS.

In certain embodiments, the supramolecular antigenic constructs of the present invention comprise an antigenic peptide sequence as described herein before, covalently attached to pegylated lysine— at least one at each terminus but particularly 1 or 2 at each terminus. The length of the PEG (polyethylenglycol) chain may vary from n=8 to n=150.000 or more, particularly from n=10 to n=80.000, more particularly from n=10 to n=10.000. In a specific embodiment of the invention the length of the PEG chain is not more than n=45, particularly between n=5 and n=40, more particularly between n=10 and n=30, and even more particularly n=10.

Liposomes that can be used in the compositions of the present invention include those known to one skilled in the art. Any of the standard lipids useful for making liposomes may be used. Standard bilayer and multi-layer liposomes may be used to make compositions of the present invention. Any method of making liposomes known to one skilled in the art may be used, including liposomes are made according to the method of Alving et al., Infect. Immun. 60:2438-2444, 1992, hereby incorporated by reference. The liposome can optionally contain an adjuvant or and immunomodulator or both. An illustrative immunomodulator is lipid A, particularly a detoxified lipid A such as, for example, monophosphoryl or diphosphoryl lipid A.

The liposome may have a dual function in that it can be used as a carrier comprising the supramolecular construct as described herein before and, at the same time, function as an adjuvant to increase or stimulate the immune response within the target patient or subject to be treated with the therapeutic vaccine according to the invention. Optionally, the liposome may, in addition, contain a further adjuvant or and immunomodulator or both such as, for example, lipid A, alum, calcium phosphate, interleukin 1, and/or microcapsules of polysaccharides and proteins, but particularly a lipid A, more particularly a detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, or alum.

The composition of the present invention comprising a supramolecular antigenic construct according to the invention and as described herein before may be prepared in the form of a liquid solution, or of an injectable suspension, or else in a solid form suitable for solubilization prior to injection in the context of, for example, a kit for making use of the present composition, as described below.

In certain embodiments, the supramolecular antigenic constructs comprise a peptide having the amino acid sequence of the SARS-CoV-2 peptide. The peptides may also comprise or correspond to whole SARS-CoV-2 surface glycoprotein peptide and active fragments thereof. Additionally, peptides useful for the present invention further comprise SARS-CoV-2 s1 subunit surface glycoprotein.

Coronaviruses

Coronaviruses (CoVs) are members of the family Coronaviridae, including betacoronavirus and alphacoronavirus— respiratory pathogens that have relatively recently become known to invade humans. The Coronaviridae family includes such betacoronavirus as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome— Corona Virus (MERS-CoV), HCoV-HKLH, and HCoV-OC43. Alphacoronavirus includes, e.g., HCoV-NL63 and HCoV-229E.

Coronaviruses invade cells through "spike” surface glycoprotein that is responsible for viral recognition of Angiotensin Converting Enzyme 2 (ACE2), a transmembrane receptor on mammalian hosts that facilitate viral entrance into host cells. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin, Nature 2020.

Symptoms associated with coronavirus-related infections include, but are not limited to, fever, tiredness, dry cough, aches and pains, shortness of breath and other breathing difficulties, diarrhea, upper respiratory symptoms (e.g. sneezing, runny nose, nasal congestion, cough, sore throat), and/or pneumonia.

Coronavirus infection 2019 (COVID-19), caused by SARS-CoV-2 (e.g., 2019-nCoV), is a disease thought to be originated from the bat. COVID-19 causes severe respiratory distress and this RNA virus strain has been the cause of a worldwide outbreak that was declared a major threat to public health and worldwide emergency. Phylogenetic analysis of the complete genome of 2019-nCoV revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, Feb 3, 2020. 2019-nCoV is thought to spread from person-to-person and the spread may be possible from contact with infected surfaces or objects.

In various embodiments, the SARS-CoV-2 surface glycoprotein comprises the amino acid sequence of SEQ ID NO: 2. In various embodiments, the SARS-CoV-2 fragment comprises amino acid residues F486, N487, Q493, Q498, T500, N501 of the SARS-CoV-2 surface glycoprotein, having the amino acid sequence of SEQ ID NO: 2, that interact with the a1 helix of the ACE2 receptor.

In some embodiments, the SARS-CoV-2 peptide is a fragment of the SARS-CoV-2 RBD or the spike protein, including the wild type or a variant (also referred to as lineages). In some embodiments, the SARS-CoV-2 peptide is a fragment of SEQ ID NO: 2 or a variant thereof. In embodiments, the wild type SARS-CoV-2 coronavirus is the "Wuhan strain.”

In embodiments, the present vaccine is pan-antigenic, thus providing immune response to the wild type (e.g., "Wuhan strain”) and numerous variants of the coronavirus. In embodiments, the present vaccine comprises one or more peptides of the wild type and/or a variants of the spike proteins, or RBD thereof. Accordingly, in various embodiments, the vaccine includes two or more peptides of a respective variant, lineage, or strain of a coronavirus protein. For example, the variants can include a coronavirus protein having a mutation (e.g., without limitation, a substitution, deletion, or insertion) in any part of the spike, or the RBD thereof, protein, such as in the S1 subunit (e.g., in the RBD of the Spike protein), or in the S2 subunit. In some embodiments, a mutation is in a glycosylation site of the Spike protein.

In some embodiments, the variants (also referred to as lineages) is one or more of B.1.1.7, B1.351, B.1, B.1.1.28, B.1.2, CAL.20C, B.6, P.1, and P.2 variants and/or any other variants, or antigenic fragments thereof. In some embodiments, the lineages include A.1, A.2, A.3, A.4, A.5, A.6, A.7, A.8, A.9, B, B.1, B.1.1, B.1.1.1, B.2, B.3, B.4, B.5, B.6, B.7, B.9, B.10, B.11, B.12, B.13, B.14, B.15, B.16, B.17, B.18, B.19, B.20, B.21, B.22, B.23, B.24, B.25, B.26, B.27, C.1, C.2, C.3, D.1, and D2.

In some embodiments, a variant is a SARS-CoV-2 protein having a variation in a glycosylation site of a Spike protein.

In some embodiments, a variant is a Spike protein having one or more of D614G, E484K, N501Y, K417N, S477G, and S477N mutations relative to the amino acid sequence of SEQ ID NO: 2 or an antigenic fragment thereof.

In some embodiments, a variant is a Spike protein having a mutation in the RBD of the Spike protein. In some embodiments, the mutation in the RBD of the Spike protein is a mutation in a glycosylation site in the RBD.

In some embodiments, a variant is a Spike protein having a mutation outside the RBD of the Spike protein.

Methods of Prevention and/or Mitigation

In another embodiment of the invention, a method for the prevention and/or treatment and/or mitigation of a coronavirus-related infection or condition is provided according to the invention and as described herein, wherein administration of said vaccine composition to a patient or subject suffering from a coronavirus-related infection results in ablation and/or mitigation of the infection.

Specifically, the present invention contemplates a method for the prevention and/or treatment and/or mitigation of a coronavirus-related infection by administering an antigenic SARS-CoV-2 fragment that interacts with the ACE2 receptor. In particular, in various embodiments, the SARS-CoV-2 fragment interacts with the ACE2 receptor.

In some embodiments, the ACE2 receptor interacts with SARS-CoV spike glycoprotein (S protein), optionally the S1 subunit, optionally the RBD of the S1 subunit. In specific embodiments, the ACE2 receptor fragment interacts with amino acid residues F486, N487, Q493, Q498, T500, N501 of the SARS-CoV-2 surface glycoprotein, having the amino acid sequence of SEQ ID NO: 2.

In various embodiments, administration of the antigenic composition results in disruption or reduction in the interaction between the ACE2 receptor and the coronavirus. For example, administration of the antigenic composition can result in disruption or reduction in SARS-CoV-2 receptor recognition and/or SARS-CoV-2 membrane fusion with a host cell. In additional embodiments, administration of the antigenic composition results in disruption or reduction in SARS-CoV- 2 infection of a host cell.

In some embodiments, the coronavirus is selected from one of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV- OC43 or the coronavirus-related infection is selected from infection by one of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome-Corona Virus (MERS-CoV), HCoV- HKU1, and HCoV-OC43. In specific embodiments, the coronavirus is SARS-CoV-2 or the coronavirus-related infection is infection by SARS-CoV. In other embodiments, the coronavirus-related infection is selected from the group consisting of coronavirus disease 19 (COVID-19) and severe acute respiratory syndrome (SARS). In an embodiment, the coronavirus-related infection is COVID-19.

In some embodiments, administration of the antigenic composition to a patient suffering from a coronavirus-related infection leads to a reduction or mitigation of symptoms associated with the coronavirus-related infection such as, but not limited to, fever, tiredness, dry cough, aches and pains, shortness of breath and other breathing difficulties, diarrhea, upper respiratory symptoms (e.g. sneezing, runny nose, nasal congestion, cough, sore throat), and/or pneumonia.

In still another embodiment, a method for the treatment of a coronavirus-related infection or condition is provided comprising administering to a patient or subject, but especially to a human at risk of suffering from such an infection or suffering from such an infection or condition, a therapeutic vaccine composition comprising an antigenic construct according to the invention.

In still another embodiment of the invention a method is provided for the preparation of a medicament for the prevention and/or treatment of a coronavirus-related infection or condition comprising using a vaccine composition according to the invention and as described herein before.

The composition of the present invention comprising a supramolecular antigenic construct is administered to a human or animal suffering from a coronavirus-related infection to induce an immune response in said human or animal to alleviate and/or mitigate symptoms associated with the infection or to restore a condition found in healthy individuals which are unaffected by the infection.

The compositions of the present invention are administered to a human or animal by any appropriate standard routes of administration. In general, the composition may be administered by topical, oral, rectal, nasal or parenteral (for example, intravenous, subcutaneous, or intramuscular) routes. In addition, the composition may be incorporated into sustained release matrices such as biodegradable polymers, the polymers being implanted in the vicinity of where delivery is desired, for example, at the site of a tumor. The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and sustained administration for a predetermined period of time.

In particular, the antigenic peptide composition according to the invention is administered by parenteral, particularly by intra-peritoneal, intravenous, subcutaneous and intramuscular injection.

The dosage of the composition will depend on the condition being treated, the particular composition used, and other clinical factors such as weight, size and condition of the patient, body surface area, the particular compound or composition to be administered, other drugs being administered concurrently, and the route of administration.

For example, the supramolecular antigenic construct compositions according to the invention may be administered parenterally, but particularly intra-peritoneally, intra-veneously, subcutaneously and intra-muscularly in a range of approximately 1.0 g to 10.0 mg per patient, though this range is not intended to be limiting. The actual amount of the composition required to elicit an immune response will vary for each individual patient depending on the immunogenicity of the composition administered and on the immune response of the individual. Consequently, the specific amount administered to an individual will be determined by routine experimentation and based upon the training and experience of one skilled in the art.

Combination Therapy

The therapeutic vaccine composition according to the invention may be administered in combination with other biologically active substances and procedures for the treatment of infections or diseases. The other biologically active substances may be part of the same composition already comprising the therapeutic vaccine according to the invention, in form of a mixture, wherein the therapeutic vaccine and the other biologically active substance are intermixed in or with the same pharmaceutically acceptable solvent and/or carrier or may be provided separately as part of a separate compositions, which may be offered separately or together in form a kit of parts.

The therapeutic vaccine composition according to the invention may be administered concomitantly with the other biologically active substance or substances, intermittently or sequentially. For example, the therapeutic vaccine composition according to the invention may be administered simultaneously with a first additional biologically active substance or sequentially after or before administration of the therapeutic vaccine. If an application scheme is chosen where more than one additional biologically active substance are administered together with the at least one therapeutic vaccine according to the invention, the compounds or substances may partially be administered simultaneously, partially sequentially in various combinations.

It is another object of the present invention to provide for mixtures of a therapeutic vaccine according to the invention and, optionally, one or more further biologically active substances, as well as to methods of using a therapeutic vaccine according to the invention, or mixtures thereof including compositions comprising said therapeutic vaccine or mixtures of therapeutic vaccines for the prevention and/or therapeutic treatment and/or alleviation of the effects of coronavirus- related infections.

In various embodiments, the composition of the present invention is co-administered in conjunction with additional therapeutic agent(s). Co-administration can be simultaneous or sequential.

In some embodiments, the additional therapeutic agent is an agent that is used to provide relief to symptoms of coronavirus infections. Such agents include remdesivir; favipiravir; galidesivir; prezcobix; lopinavir and/or ritonavir and/or arbidol; mRNA-1273; MSCs-derived exosomes; lopinavir/ritonavir and/or ribavirin and/or IFN-beta; xiyanping; anti-VEGF-A (e.g. Bevacizumab); fingolimod; carrimycin; hydroxychloroquine; darunavir and cobicistat; methylprednisolone; brilacidin; leronlimab (PRO 140); and thalidomide.

In some embodiments, the additional therapeutic agent is chloroquine, including chloroquine phosphate.

In an embodiment, the additional therapeutic agent is a composition comprising one or more HIV drugs. In some embodiments, the composition comprises a combination of one or more of lopinavir and/or ritonavir and/or arbidol.

The other additional therapeutic agent or compound may exert its biological effect by the same or a similar mechanism as the therapeutic vaccine according to the invention or by an unrelated mechanism of action or by a multiplicity of related and/or unrelated mechanisms of action.

In a specific embodiment of the invention, the compositions and mixtures according to the invention and as described herein before comprise the vaccine according to the invention and the additional therapeutic agent, respectively, in a therapeutically or prophylactically effective amount.

In some embodiments, a combination of the present vaccine and other vaccines may boost immunity in certain types of patients, including elderly patients, patents with comorbidities, and patients with compromised immune systems. In some embodiments, the present vaccine enhances the efficacy of other vaccines, e.g. other coronavirus vaccines, e.g. a coronavirus vaccine which comprises one or more of a live attenuated virus, an inactivated virus, a non-replicating viral vector, a replicating viral vector, a recombinant protein, a peptide, a virus-like particle, DNA, RNA, mRNA, another macromolecule, and a fragment thereof. In some embodiments, the coronavirus vaccine is selected from mRNA-1273, AZD1222, BNT162, Ad5-nCoV, INO-4800, and LV-SMENP-DC, and pathogen-specific aAPC, or a variant or derivative thereof. In some embodiments, the coronavirus vaccine comprises an mRNA vaccine encoding SARS-CoV-2 spike (S) protein, optionally LNP-encapsulated, like mRNA-1273. In some embodiments, the coronavirus vaccine comprises a viral vector vaccine expressing the S protein, optionally a viral vector (ChAdOxl - chimpanzee adenovirus Oxford 1) vaccine (ChAdOxl nCoV-19) expressing the S protein, like AZD1222. In some embodiments, the coronavirus vaccine comprises an mRNA vaccine encoding an optimized SARS-CoV-2 RBD, like BNT162b1. In some embodiments, the coronavirus vaccine comprises an mRNA vaccine encoding an optimized full-length S protein, like BNT162b2. In some embodiments, the coronavirus vaccine comprises Adenovirus type 5 vector that expresses a protein selected from spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsid phosphoprotein N; optionally Adenovirus type 5 vector that expresses S protein, like Ad5-nCoV. In some embodiments, the coronavirus vaccine comprises a plasmid encoding S protein delivered by electroporation, optionally a DNA plasmid encoding S protein delivered by electroporation, like I NO-4800. In some embodiments, the coronavirus vaccine comprises dendritic cells (DCs) modified with lentiviral vector expressing synthetic minigene based on domains of selected viral proteins, administered with antigen-specific cytotoxic T lymphocytes (CTLs), like LV-SMENP-DC. In some embodiments, the coronavirus vaccine comprises artificial antigen-presenting cells (aAPCs) modified with lentiviral vector expressing synthetic minigene based on domains of selected viral proteins, like pathogen-specific aAPC.

The compositions of the present invention are administered to a human or animal by any appropriate means, e.g. by injection. For example, a modified antigenic peptide reconstituted in liposomes is administered by subcutaneous injection. Whether internally produced or provided from external sources, the circulating antibodies bind to antigen and reduce or inactivate its ability to stimulate disease.

Methods of Making

In another aspect, the present invention contemplates methods of producing antibodies against the antigenic composition described herein, comprising administering an antigen sample into an animal and recovering the animal's serum comprising antigen-specific antibodies. In some embodiments, the antigen sample comprises a SARS-CoV-2 fragment that interacts with the ACE2 receptor.

The lipophilic or hydrophobic moiety according to the present invention may be a fatty acid, a triglyceride or a phospholipid wherein the fatty acid carbon backbone has at least 10 carbon atoms. Particularly, the lipophilic or hydrophobic moiety is a fatty acid with a carbon backbone of at least approximately 14 carbon atoms and up to approximately 24 carbon atoms, with each individual number of carbon atom falling within this range also being part of the present invention. More particularly, the lipophilic or hydrophobic moiety has a carbon backbone of at least 14 carbon atoms, but especially 16 carbon atoms. Examples of hydrophobic moieties include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid. In a specific embodiment of the present invention the lipophilic or hydrophobic moiety is palmitic acid.

Liposomes may also be prepared by the crossflow injection technique as described, for example, in Wagner et al (2002) Journal of Liposome Research Vol 12(3), pp 259-270. During the injection of lipid solutions into an aqueous buffer system, lipids tend to form "precipitates", followed by self-arrangement in vesicles. The obtained vesicle size depends on factors such as lipid concentration, stirring rate, injection rate, and the choice of lipids. The preparation system may consist of a crossflow injection module, vessels for the polar phase (e.g. a PBS buffer solution), an ethanol/lipid solution vessel and a pressure device, but particularly a nitrogen pressure device. While the aqueous or polar solution is pumped through the crossflow injection module the ethanol/lipid solution is injected into the polar phase with varying pressures applied.

For determining immunogenicity of the modified SARS-CoV-2 antigenic construct a suitable animal selected from the group consisting of mice, rats, rabbits, pigs, birds, etc, but particularly mice, especially C57BL/6 mice are immunized with the antigenic peptide. Immunogenicity of the antigenic construct is determined by probing Sera samples in suitable time intervals after immunization using a immunoassay such as, for example, an ELISA assay.

The modified antigenic construct, particularly the palmitoylated antigenic construct and, more particularly, the palmitoylated SARS-CoV-2 peptide construct is used for the immunization of an animal, particularly a mammal or a human, suffering from symptoms associated with a coronavirus-related infection.

The supramolecular antigenic construct according to the present invention, but particularly a vaccine composition comprising such a supramolecular antigenic construct according to the invention is administered to an animal, particularly a mammal or a human, by any appropriate standard routes of administration. In general, the composition may be administered by topical, oral, rectal, nasal or parenteral (for example, intravenous, subcutaneous, or intramuscular) routes. In addition, the composition may be incorporated into sustained release matrices such as biodegradable polymers, the polymers being implanted in the vicinity of where delivery is desired, for example, at the site of a tumor. The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and sustained administration for a predetermined period of time.

In a specific embodiment of the invention the antigenic construct according to the invention, particularly a vaccine composition comprising said antigenic construct in a pharmaceutically acceptable form, is administered in repeated doses, in particular in 1 to 15 doses, more particularly in 2 to 10 doses, more particularly in 3 to 7 doses and even more particularly in 4 to 6 doses, in time intervals of between 1 and 10 weeks, particularly in time intervals of between 1 and 6 weeks, more particularly in time intervals of between 1 and 4 weeks, and even more particularly in time intervals of between 2 and 3 weeks. The immune response is monitored by taking Sera samples at a suitable time after boosting, particularly 3 to 10 days after boosting, more particularly 4 to 8 days after boosting and more particularly 5 to 6 days after boosting and determining the immunogenicity of the antigenic construct using known methodology, particularly one of the commonly used immunoassays such as, for example, an ELISA assay.

Immunization with the antigenic construct according to the invention, but particularly with a vaccine composition comprising the antigenic construct according to the invention in a pharmaceutically acceptable form leads to a significant and highly specific immune response in the treated animal or human.

The compositions of the present invention may also be used to produce antibodies directed against antigenic peptides.

In embodiments, the administration of the antigenic composition results in the production of conformationally specific antibodies against SARS-CoV-2 S1 RBD, optionally in a beta-sheet interacting region. In embodiments, the antibodies are administered to individuals to passively immunize them against a variety of coronavirus-related infections or conditions, including but not limited to, COVID-19 and SARS.

Subjects

In illustrative embodiments, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).

In various embodiments, the mammal is a human. In some embodiments, the human is an adult aged 18 years or older. In some embodiments, the human is a child aged 17 years or less. In an embodiment, the subject is male, e.g., a male human. In another embodiment, the subject is a female subject. In illustrative embodiments, the subject is a female subject, e.g., a female human.

Kjts

Kits comprising a composition comprising any one of the foregoing of the present invention are also provided.

The invention provides kits that can simplify the administration of any agent described herein. An illustrative kit of the invention comprises any composition described herein in unit dosage form. In one embodiment, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or illustrative language (e.g., "such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Variations of described embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Definitions

The use of the terms "a” and "an” and "the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising,” "having,” "including,” and "containing” are to be construed as open-ended terms (i.e., meaning "including, but not limited to,”) unless otherwise noted.

The terms "polypeptide", "peptide", and "protein", as used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.

The term "peptides," are chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. As such, the term "amino terminus" (abbreviated N-terminus) refers to the free alpha-amino group on the amino acid at the amino terminal of the peptide, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term "carboxy terminus" (abbreviated C-terminus) refers to the free carboxyl group on the amino acid at the carboxy terminus of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide.

Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminal and increasing in the direction toward the carboxy terminal of the peptide. Thus, when one amino acid is said to "follow" another, that amino acid is positioned closer to the carboxy terminal of the peptide than the preceding amino acid.

The term "residue" is used herein to refer to an amino acid that is incorporated into a peptide by an amide bond. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art. The phrase "consisting essentially of" is used herein to exclude any elements that would substantially alter the essential properties of the peptides to which the phrase refers. Thus, the description of a peptide "consisting essentially of " excludes any amino acid substitutions, additions, or deletions that would substantially alter the biological activity of that peptide.

Furthermore, one of skill will recognize that, as mentioned above, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The phrases "isolated" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. Thus, the peptides described herein do not contain materials normally associated with their in situ environment. Typically, the isolated, immunogenic peptides described herein are at least about 80% pure, usually at least about 90%, or at least about 95% as measured by band intensity on a silver stained gel.

Protein purity or homogeneity may be indicated by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.

When the immunogenic peptides are relatively short in length (i.e., less than about 50 amino acids), they are often synthesized using standard chemical peptide synthesis

Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a method for the chemical synthesis of the immunogenic peptides described herein. Techniques for solid phase synthesis are known to those skilled in the art. Alternatively, the immunogenic peptides described herein are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the peptide, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the peptide in a host, isolating the expressed peptide or polypeptide and, if required, renaturing the peptide. Techniques sufficient to guide one of skill through such procedures are found in the literature.

Once expressed, recombinant peptides can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Substantially pure compositions of about 50% to 95% homogeneity are provided, and 80% to 95% or greater homogeneity are also provided for use as therapeutic agents.

One of skill in the art will recognize that after chemical synthesis, biological expression or purification, the immunogenic peptides may possess a conformation substantially different than the native conformations of the constituent peptides. In this case, it is often necessary to denature and reduce the antiproliferative peptide and then to cause the peptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art.

Antigenicity of the purified protein may be confirmed, for example, by demonstrating reaction with immune serum, or with antisera produced against the protein itself.

The terms "a", "an" and "the" as used herein are defined to mean "one or more" and include the plural unless the context is inappropriate.

The terms "detecting" or "detected" as used herein mean using known techniques for detection of biologic molecules such as immunochemical or histological methods and refer to qualitatively or quantitatively determining the presence or concentration of the biomolecule under investigation.

By "isolated" is meant a biological molecule free from at least some of the components with which it naturally occurs.

The terms "antibody" or "antibodies" as used herein is an art recognized term and is understood to refer to molecules or active fragments of molecules that bind to known antigens, particularly to immunoglobulin molecules and to immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that immunospecifically binds an antigen. The immunoglobulin according to the invention can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2) or subclasses of immunoglobulin molecule.

"Antibodies" are intended within the scope of the present invention to include monoclonal antibodies, polyclonal, chimeric, single chain, bispecific, simianized, human and humanized antibodies as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab and F(ab') 2 fragments, including the products of an Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above. These active fragments can be derived from an antibody of the present invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121 :663-69, Academic Press, 1986.

A "humanized antibody" refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity. Methods to obtain "humanized antibodies" are well known to those skilled in the art. (see, e.g., Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio/Technology, 9:421 (1991)).

A "humanized antibody" may also be obtained by a novel genetic engineering approach that enables production of affinity-matured humanlike polyclonal antibodies in large animals such as, for example, rabbits.

The term "monoclonal antibody" is also well recognized in the art and refers to an antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically made by fusing a normally short-lived, antibody-producing B cell to a fast-growing cell, such as a cancer cell (e.g. an immortal cell). The resulting hybrid cell, or hybridoma, multiplies rapidly, creating a clone that produces large quantities of the antibody.

"Functionally equivalent antibody" is understood within the scope of the present invention to refer to an antibody which substantially shares at least one major functional property with an antibody mentioned above and herein described comprising: binding specificity to the SARS-CoV-2 protein.

The term "antigen" refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions responsible for antigenicity or antigenic determinants.

As used herein, the term "soluble" means partially or completely dissolved in an aqueous solution.

Also as used herein, the term "immunogenic" refers to substances which elicit or enhance the production of antibodies, T-cells and other reactive immune cells directed against an immunogenic agent and contribute to an immune response in humans or animals.

An immune response occurs when an individual produces sufficient antibodies, T-cells and other reactive immune cells against administered immunogenic compositions of the present invention to moderate or alleviate the disorder to be treated. The term "hybridoma" is art recognized and is understood by those of ordinary skill in the art to refer to a cell produced by the fusion of an antibody-producing cell and an immortal cell, e.g. a multiple myeloma cell. This hybrid cell is capable of producing a continuous supply of antibody. See the definition of "monoclonal antibody" above and the Examples below for a more detailed description of the method of fusion.

The term "carrier" as used herein means a structure in which antigenic peptide or supramolecular construct can be incorporated into or can be associated with, thereby presenting or exposing antigenic peptides or part of the peptide to the immune system of a human or animal. Any particle that can be suitably used in animal or human therapy such as, for example, a vesicle, a particle or a particulate body may be used as a carrier within the context of the present invention.

The term "carrier" further comprises methods of delivery wherein supramolecular antigenic construct compositions comprising the antigenic peptide may be transported to desired sites by delivery mechanisms. One example of such a delivery system utilizes colloidal metals such as colloidal gold.

EXAMPLES

Example 1: SARS-CoV-2 Surrogate Virus Neutralization Test

A SARS-CoV-2 surrogate virus neutralization test was performed using a SARS-CoV-2 surrogate virus neutralization test kit (GenScript). The test detects neutralizing antibodies against SARS-CoV-2 that are able to block the interaction between the receptor binding domain of the viral spike glycoprotein (RBD) with the ACE2 cell surface receptor. The assay detects antibodies that neutralize the RBD-ACE2 interaction in serum samples. The test is both species and isotype independent.

The SARS-CoV-2 surrogate virus neutralization test is a blocking ELISA assay, which mimics the virus neutralization process measuring protein-protein interaction between horseradish peroxidase (HRP)-RBD and hACE2 and possible reduction of this interaction by neutralizing antibodies against the SARS-CoV-2 RBD.

First, the samples and controls were pre-incubated with the HRP-RBD to allow the binding of the circulating neutralization antibodies to HRP-RBD. The mixture was then added to the capture plate, which was pre-coated with the hACE2 protein. The unbound HRP-RBD, as well as any HRP-RBD bound to non-neutralizing antibody, were captured on the plate, while the circulating neutralization antibodies-HRP-RBD complexes remained in the supernatant and were removed during washing. After washing steps were complete, a tetramethylbenzidine (TMB) solution was added. The reaction was then quenched by adding a Stop Solution, and the absorbance of the samples were read at 450nm and were inversely dependent on the titer of the anti-SARS-CoV-2 neutralizing antibodies. Neutralization Reaction Procedure

In separate tubes, the diluted Positive Control, diluted Negative Control, and the test samples were mixed with the diluted HRP-RBD solution with a volume ratio of 1 :1 followed by an incubation at 37°C for 30 minutes. 100 pL each of the positive control mixture, the negative control mixture, and the sample mixture was added to the corresponding wells in the 96 well plate. After an incubation at 37°C for 15 minutes, the plate was washed with 260 pL of 1 x Wash Solution for four times and any residual liquid in the wells was removed after washing steps.

Substrate Reaction and Absorbance Measurement

100 pL of TMB Solution was added to each well and the plate was incubated in the dark at 20 - 25°C for 15 minutes. The reaction was quenched with 50 pL of Stop Solution added to each well. Absorbance was determined immediately in the microtiter plate reader at 450 nm (BertholdTech TriStar2S).

The serum samples were then tested:

1. Antiserum rabbit 1, not purified (A: antigen) - undiluted - "Anti-ALS A -1,” in which the sample A: antigen is a SARS-CoV-2 peptide, a fragment of the SARS-CoV-2 RBD, specifically comprising amino acid residues 485-502 of the SARS-CoV-2 surface glycoprotein. The SARS-CoV-2 peptide has the amino acid sequence of SEQ ID NO: 3.

2. Antiserum rabbit 2, not purified (A: antigen) - undiluted - "Anti-ALS A -2,” in which the sample A: antigen is a SARS- CoV-2 peptide, a fragment of the SARS-CoV-2 RBD, specifically comprising amino acid residues 485-502 of the SARS- CoV-2 surface glycoprotein. The SARS-CoV-2 peptide has the amino acid sequence of SEQ ID NO: 3.

3. Antiserum rabbit , not purified (B: control) - undiluted - "Anti-ALS -B”

4. Preimmune serum rabbit 1 day 0 (A: antigen) - undiluted - "Pre-ALS A -1”

5. Preimmune serum rabbit 2 day 0 (A: antigen) - undiluted - "Pre-ALS A -2”

6. Preimmune serum rabbit day 0 (B: control) - undiluted - "Pre-ALS-B”

7. Antiserum pooled rabbit 1&2, Affinity purified from antigen/lipo (0.42 mg/mL) -"Anti- ALS 1&2 purif - in the following concentrations: 10 ng/ L, 20 ng/ L, 30 ng/ L, 40 ng/ L, 80 ng/ L. The sample antigen is a SARS-CoV-2 peptide, a fragment of the SARS-CoV-2 RBD, specifically comprising amino acid residues 485-502 of the SARS-CoV- 2 surface glycoprotein. The SARS-CoV-2 peptide has the amino acid sequence of SEQ ID NO: 3.

The results of the SARS-CoV-2 Surrogate Virus Neutralization experiment (e.g., inhibition percentages) are depicted in Figure 2 and Table 1 below. Figure 3 depicts the percent inhibition relative to the positive control of the test of the "Anti- ALS 1&2 purif group in concentrations of 10 ng/ L, 20 ng/ L, 30 ng/ L, 40 ng/ L, 80 ng/ L. Percent inhibition is calculated as 1 - ABS value of Sample / ABS value of Negative Control) x 100%. Table 1. Inhibition percentages captured from the SARS-CoV-2 Surrogate Virus Neutralization experiment.

Figure 4 shows percent inhibition of Anti-ALS1, Anti-ALS2, Anti-ALSB, Pre-ALS1, Pre-ALS2, and Pre-ALSB, each diluted 1 :9 with sample buffer. Figure 5 depicts percent inhibition of BLCO serum dilutions. Figure 6 shows percent inhibition of the "ALS-B” group and the Non-immunized group in the concentration of 100 ng/pL.

Example 2: Cytopathic Effect-Based Virus Neutralization Test Vero E6 cells (ATCC - CRL 1586) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) -supplemented with 10% of fetal bovine serum (FBS) - at 37°C, in a 5% CO2 humidified incubator. Sub-confluent cell monolayers of Vero E6 were prepared in growth medium containing 2% FBS in 96-well plates for titration and neutralization tests of SARS- CoV-2.

Then, SARS-CoV-2 was isolated on the Vero E6 cell line and the viral load was quantified via qRT-PCR in the inoculum and after 4 days in culture (or when the cytopathic effect was 50-75%). The SARS-CoV-2 isolate IAIO was selected for cytopathic effect-based VNT (see Figures 8A-B), and the copy number was quantified (PrimerDesign genesig® COVID-19 2G RT-PCR assay) for the preliminary test, in order to check the rough culture conditions. 10⁵ copies/well were used in the preliminary virus neutralization test. Figures 8A-B depict the cytopathic effects of SARS-CoV-2 exhibited in Vero E6 cell cultures when the Vero E6 cells were inoculated with oropharyngeal swab sample IAIU (10⁴ copies/ L). Figure 8A shows Vero cell cultures in negative control, and Figure 8B shows the cytopathic effects consisting of rounding, detachment of cells, blebbing and intense vacuolization, 4 days after inoculation.

SARS-CoV-2 titration and CPE-based VNT

The virus is titrated in serial 1 log dilutions (from 1 log to 12 log) to obtain a 50% tissue culture infective dose (TCID50) on 96-well culture plates of Vero E6 cells. The plates are observed daily for a total of 5 days for the presence of CPE by means of a phase-contrast optical microscope.

The end-point titers are calculated according to the Reed & Muench method based on six replicates for titration.

The preliminary CPE-based VNT is prepared after virus and serum samples are in contact 1 hour, at 37°C, and afterwards 20 mI_ of the suspension is inoculated on the first well and diluted in serial 1 log dilutions (from 1 log to 12 log) on the rest of the wells of the row of the 96 well plate. The last two rows of the plate contain the controls.

Example 3: Production of SARS-CoV-2 Peptide Antigen

In order to produce antibodies against the SARS-CoV-2 peptide antigenic composition, an antigen sample containing a SARS-CoV-2 fragment that interacts with ACE2 receptor is administered into an animal and the animal's serum comprising antigen-specific antibodies is then recovered. The SARS-CoV-2 fragment that interacts with ACE2 receptor has the amino acid sequence of SEQ ID NO: 3.

Example 4: Administration of Supramolecular Antigenic Composition Prevents and/or Mitigates SARS-CoV-2 Infection.

Upon administration of the antigenic composition comprising a SARS-CoV-2 receptor fragment to a patient suffering from SARS-CoV-2 infection (COVID-19), the symptoms associated with the infection are mitigated or ablated.

EQUIVALENTS

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Claims

CLAIMS What is claimed is:

1. A method for the prevention or mitigation of a coronavirus-related infection comprising administering to a patient at risk of suffering from such an infection or a patient suffering from such an infection an antigenic composition comprising one or more copies of a palmitoylated SARS-CoV-2 peptide antigen reconstituted in a liposome.

2. The method of claim 1, wherein the SARS-CoV-2 peptide antigen is pre-formed by on-resin standard automated peptide synthesis.

3. The method of claim 2, wherein the SARS-CoV-2 peptide antigen is modified by on-resin grafting of a palmitoyl moiety to the terminal amino acid residues of the pre-formed SARS-CoV-2 peptide.

4. The method of any one of the previous claims, wherein the SARS-CoV-2 peptide is a fragment of the SARS- CoV-2 receptor binding domain (RBD).

5. The method of any one of the previous claims, wherein the SARS-CoV-2 peptide is a fragment of the SARS- CoV-2 RBD of the SARS-CoV-2 s1 subunit surface glycoprotein.

6. The method of any one of the previous claims, wherein the SARS-CoV-2 RBD fragment interacts with the ACE2 receptor.

7. The method of claim 6, wherein the ACE2 receptor interacts with the SARS-CoV-2 RBD fragment.

8. The method of any one of the preceding claims, wherein the ACE2 receptor comprises the amino acid sequence of SEQ ID NO: 1 and/or the SARS-CoV-2 peptide is derived from the amino acid sequence of SEQ ID NO: 2

9. The method of claim 8, wherein the ACE2 receptor interacts with SARS-CoV spike glycoprotein (S protein), optionally the S1 subunit, optionally the RBD of the S1 subunit.

10. The method of claim 8 or 9, wherein the ACE2 receptor interacts with amino acid residues F486, N487, Q493, Q498, T500, N501 of the amino acid sequence of SEQ ID NO: 2.

11. The method of any one of the preceding claims, wherein the SARS-CoV-2 fragment comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

12. The method of claim 11, wherein the SARS-CoV-2 peptide antigen comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions.

13. The method of claim 11 or 12, wherein the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions comprises at least one palmitoyl moiety on the N terminus.

14. The method of any one of claims 11-13, wherein the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions comprises at least one palmitoyl moiety on the C terminus.

15. The method of any one of claims 11-14, wherein the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions comprises at least one palmitoyl moiety on the N and C terminus.

16. The method of any one of claims 11-15, wherein the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions comprises at least two palmitoyl moiety on the N and C terminus.

17. The method of any one of claims 11-16, wherein the palmitoylated SARS-CoV-2 peptide antigen is H(Pal)Lys- (Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, wherein Pal is a palmitoyl moiety or H(Pal)Lys-(Pal)Lys- SEQ ID NO: 4-(Pal)Lys- (Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, wherein Pal is a palmitoyl moiety.

18. The method of any one of claims 11-17, wherein the palmitoylated SARS-CoV-2 peptide antigen is H(Pal)Lys- (Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, wherein Pal is a palmitoyl moiety or H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, wherein Pal is a palmitoyl moiety, wherein Pal is a palmitoyl moiety.

19. The method of any one of the previous claims, wherein administration of the antigenic composition results in disruption or reduction in the interaction between the ACE2 receptor and the coronavirus.

20. The method of any one of the previous claims, wherein administration of the antigenic composition results in disruption or reduction in SARS-CoV-2 receptor recognition and/or SARS-CoV-2 membrane fusion with a host cell.

21 . The method of any one of the previous claims, wherein administration of the antigenic composition results in disruption or reduction in SARS-CoV-2 infection of a host cell.

22. The method of any one of the previous claims, wherein administration of the antigenic composition results in the production of conformationally specific antibodies against SARS-CoV-2 S1 RBD, optionally in a beta-sheet interacting region.

23. The method of claim 22, wherein the coronavirus is SARS-CoV-2 or the coronavirus-related infection is infection by SARS-CoV.

24. The method of any one of the previous claims, wherein the coronavirus-related infection is selected from the group consisting of coronavirus disease 19 (COVID-19) and severe acute respiratory syndrome (SARS).

25. The method of claim 24, wherein the coronavirus-related infection is COVID-19.

26. The method of any of the above claims, wherein administration of the antigenic composition to a patient suffering from a coronavirus-related infection leads to a reduction or mitigation of symptoms associated with the coronavirus-related infection.

27. The method of claim 26, wherein one or more of the symptoms associated with the coronavirus-related infection are selected from the group consisting of fever, tiredness, dry cough, aches and pains, shortness of breath and other breathing difficulties, diarrhea, upper respiratory symptoms (e.g. sneezing, runny nose, nasal congestion, cough, sore throat), and/or pneumonia.

28. The method of any one of the previous claims, wherein the antigenic composition is administered with an additional therapeutic agent or an additional vaccine agent.

29. The method of any one of the previous claims, wherein the antigenic composition is administered before, after, or concurrently with the additional therapeutic agent or the additional vaccine agent.

30. The method of any one of the previous claims, wherein the additional therapeutic agent is used to provide relief to symptoms of coronavirus-related infections.

31. The method of any one of the previous claims, wherein the additional therapeutic agent is selected from the group consisting of remdesivir; favipiravir; galidesivir; prezcobix; lopinavir and/or ritonavir and/or arbidol; MSCs-derived exosomes; lopinavir/ritonavir and/or ribavirin and/or IFN-beta; xiyanping; anti-VEGF-A (e.g. Bevacizumab); fingolimod; carrimycin; hydroxychloroquine; darunavir and cobicistat; methylprednisolone; brilacidin; leronlimab (PRO 140); and thalidomide.

32. The method of claim 30, wherein the additional therapeutic agent is a composition comprising one or more HIV drugs, optionally selected from the group consisting of lopinavir, ritonavir, and arbidol.

33. The method of claim 30, wherein the additional vaccine agent is a live attenuated virus, an inactivated virus, a non-replicating viral vector, a replicating viral vector, a recombinant protein, a peptide, a virus-like particle, DNA, RNA, mRNA, or another macromolecule, and a fragment thereof, optionally selected from mRNA-1273, AZD1222, BNT162, Ad5-nCoV, INO-4800, and LV-SMENP-DC.

34. A method of producing antibodies against the antigenic composition described herein, comprising administering an antigen sample into an animal and recovering the animal's serum comprising antigen-specific antibodies.

35. The method of claim 34, wherein the antigen sample comprises a SARS-CoV-2 fragment that interacts with the ACE2 receptor.

36. A method of vaccinating against a coronavirus-related infection, optionally SARS-CoV-2 infection, optionally COVID-19 comprising administering an antigenic composition described herein to a patient in need thereof.

37. An antigenic construct comprising one or more copies of a palmitoylated SARS-CoV-2 peptide antigen reconstituted in a liposome.

38. The antigenic construct of claim 37, wherein the SARS-CoV-2 peptide antigen is pre-formed by on-resin standard automated peptide synthesis and modified by on-resin grafting of a palmitoyl moiety to the terminal amino acid residues of the pre-formed SARS-CoV-2 peptide antigen.

39. The antigenic construct of claim 37 or 38, wherein the SARS-CoV-2 peptide antigen comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions.

40. The antigenic construct of claim 39, wherein the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions further comprises at least one palmitoyl moiety on the N terminus.

41 . The antigenic construct of claim 39 or 40, wherein the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions further comprises at least one palmitoyl moiety on the C terminus.

42. The antigenic construct of any one of claims 39-41, wherein the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions further comprises at least one palmitoyl moiety on the N and C terminus.

43. The antigenic construct of any one of claims 39-42, wherein the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions further comprises at least two palmitoyl moiety on the N and C terminus.

44. The antigenic construct of any one of claims 39-43, wherein the palmitoylated SARS-CoV-2 peptide antigen is H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, wherein Pal is a palmitoyl moiety or H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, optionally with about 6 or about 5, or about 4, or about 3, or about 2, or about 1 substitutions or deletions, wherein Pal is a palmitoyl moiety.

45. The antigenic construct of any one of claims 39-44, wherein the palmitoylated SARS-CoV-2 peptide antigen is H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, wherein Pal is a palmitoyl moiety or H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, wherein Pal is a palmitoyl moiety.

46. A method for the prevention of a coronavirus-related infection comprising administering to a patient at risk of suffering from such an infection an antigenic composition comprising one or more copies of a palmitoylated SARS-

CoV-2 peptide antigen reconstituted in a liposome, the a palmitoylated SARS-CoV-2 peptide antigen comprising an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

47. The method of claim 46, wherein the palmitoylated SARS-CoV-2 peptide antigen is:

H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, wherein Pal is a palmitoyl moiety or H(Pal)Lys-(Pal)Lys- SEQ ID NO: 3-(Pal)Lys-(Pal)Lys-OH, wherein Pal is a palmitoyl moiety.