DESIGNER PEPTIDES AND PROTEINS FOR THE DETECTION, PREVENTION AND TREATMENT OF CORONAVIRUS DISEASE, 2019 (COVID-19)
The present disclosure is directed to a relief system for the effective detection, prevention, and treatment of COVID-19, including (1) serological diagnostic assays for the detection of viral infection and epidemiological surveillance, (2) high-precision, site-directed peptide immunogen constructs for the prevention of infection by SARS-CoV-2, (3) receptor-based antiviral therapies for the treatment of the disease in infected patients, and (4) designer protein vaccine containing S1-RBD-sFc. The disclosed relief system utilizes amino acid sequences from SARS-CoV-2 proteins as well as human receptors for the design and manufacture of optimal SARS-CoV-2 antigenic peptides, peptide immunogen constructs, CHO-derived protein immunogen constructs, long-acting CHO-derived ACE2 proteins, and formulations thereof, as diagnostics, vaccines, and antiviral therapies for the detection, prevention, and treatment of COVID-19.
1 . A fusion protein comprising:
a) an amino acid sequence derived from the receptor binding domain (RBD) of the Spike (S) protein from SARS-CoV-2 selected from the group consisting of SEQ ID NO: 225 and SEQ ID NO: 226;
b) an optional hinge region from an IgG molecule selected from the group consisting of SEQ ID NO: 166-225; and
c) an Fc fragment of an IgG molecule selected from the group consisting of SEQ ID NOs: 231-234,
wherein the amino acid sequence in (a) is covalently linked to the Fc fragment in (c) through directly or through the optional hinge region in (b).
2 . The fusion protein according to claim 1 , wherein
the Fc fragment in (c) has an amino acid sequence of SEQ ID NO: 232 and
the optional hinge region has an amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 188.
3 . The fusion protein according to claim 1 , wherein the fusion protein is selected from the group consisting of: S1-RBD-sFc of SEQ ID NOs: 235, S1-RBDa-sFc of SEQ ID NO: 236, and S1-RBD-Fc of SEQ ID NO: 355.
4 . A COVID-19 vaccine composition comprising:
a) the fusion protein according to claim 3 ; and
b) a pharmaceutically acceptable excipient.
5 . The COVID-19 vaccine composition according to claim 4 , wherein the fusion protein is S1-RBD-sFc of SEQ ID NO: 235.
6 . The COVID-19 vaccine composition according to claim 4 further comprising a Th/CTL peptide.
7 . The COVID-19 vaccine composition according to claim 6 , wherein the Th/CTL peptide is derived from the SARS-CoV-2 M protein of SEQ ID NO: 1, the SARS-CoV-2 N protein of SEQ ID NO: 6, the SARS-CoV-2 S protein of SEQ ID NO: 20, a pathogen protein, or any combination thereof.
8 . The COVID-19 vaccine composition according to claim 7 , wherein
a. the Th/CTL peptide derived from the SARS-CoV-2 M protein is SEQ ID NO: 361;
b. the Th/CTL peptide derived from the SARS-CoV-2 N protein is selected from the group consisting of SEQ ID NOs: 9-16, 19, 153-160, 165, 347, 350, 351, and 363;
c. the Th/CTL peptide derived from the SARS-CoV-2 S protein is selected from the group consisting of SEQ ID NOs: 35-36, 39-48, 145-152, 161-164, 345-346, 348, 362, 364, and 365;
d. the Th/CTL peptide derived from a pathogen protein is selected from the group consisting of SEQ ID NOs: 49-100.
9 . The COVID-19 vaccine composition according to claim 4 , further comprising a mixture of Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66.
10 . The COVID-19 vaccine composition according to claim 9 , wherein each of the Th/CTL peptides are present in the mixture in equal-weight amounts.
11 . The COVID-19 vaccine composition according to claim 10 , wherein the ration (w:w) of the S1-RBD-sFc protein to the total weight of the mixture of Th/CTL peptides is 88:12.
12 . The COVID-19 vaccine composition according to claim 4 , wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.
13 . The COVID-19 vaccine composition according to claim 4 , wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, aluminum phosphate, histidine, histidine HCl.H 2 O, arginine HCl, polyoxyethylene (20) sorbitan monooleate, hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof.
14 . A COVID-19 vaccine composition comprising:
a. a S1-RBD-sFc protein of SEQ ID NO: 235;
b. a Th/CTL peptide selected from the group consisting of SEQ ID NOs: 9-16, 19, 35-36, 39-100, 145-165, 345-348, 350, 351, 362-365, and any combination thereof;
c. a pharmaceutically acceptable excipient.
15 . The COVID-19 vaccine composition according to claim 14 , wherein the Th/CTL peptides in (b) is a mixture of SEQ ID NOs: 345, 346, 347, 348, 361, and 66.
16 . The COVID-19 vaccine composition according to claim 15 , wherein each of the Th/CTL peptides are present in the mixture in equal-weight amounts.
17 . The COVID-19 vaccine composition according to claim 16 , wherein the ration (w:w) of the S1-RBD-sFc protein to the total weight of the mixture of Th/CTL peptides is 88:12.
18 . The COVID-19 vaccine composition according to claim 14 , wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.
19 . The COVID-19 vaccine composition according to claim 14 , wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, aluminum phosphate, histidine, histidine HCl.H 2 O, arginine HCl, polyoxyethylene (20) sorbitan monooleate, hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof.
20 . The COVID-19 vaccine composition according to claim 14 , wherein
the Th/CTL peptide is a mixture of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, wherein each peptide is present in the mixture in equal-weight amounts;
the pharmaceutically acceptable excipient is a combination of a CpG1 oligonucleotide, aluminum phosphate, histidine, histidine HCl.H 2 O, arginine HCl, polyoxyethylene (20) sorbitan monooleate, hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.
21 . The COVID-19 vaccine composition according to claim 20 , wherein the total amount of the S1-RBD-sFc protein of SEQ ID NO: 235 is between about 10 μg to about 200 μg; and
the total amount of the Th/CTL peptides is between about 2μg to about 25 μg.
22 . (canceled)
23 . (canceled)
24 . (canceled)
25 . A method for preventing COVID-19 in a subject comprising administering a pharmaceutically effective amount of the vaccine composition according to claim 12 to a subject.
26 . The method according to claim 25 , wherein the pharmaceutically effective amount of the vaccine composition is administered to the subject in two doses.
27 . (canceled)
28 . A method for generating antibodies against SARS-CoV-2 comprising administering a pharmaceutically effective amount of the vaccine composition according to claim 14 to a subject.
29 . (canceled)
30 . (canceled)
31 . (canceled)
32 . (canceled)
33 . (canceled)
34 . (canceled)
35 . (canceled)
36 . (canceled)
37 . (canceled)
38 . (canceled)
39 . (canceled)
40 . A method for detecting infection by SARS-CoV-2 comprising:
a) attaching an antigenic peptide selected from the group consisting of SEQ ID NOs: 4-5, 17-18, 23-24, 26, 29-34, 37-38, 259, 261, 263, 265, 266, 270, 281, 308, 321, 322, 323, and 324 and any combination thereof to a solid support,
b) exposing the antigenic peptide attached to the solid support in (a) to a biological sample containing antibodies from a patient, under conditions conducive to binding of the antibody to the peptide, and
c) detecting the presence of antibodies bound to the peptide attached to the solid support in (b).
41 . The method according to claim 40 , wherein the antigenic peptide of (a) is selected from the group consisting of SEQ ID NOs: 5, 18, 38, 261, 266, 281, 322, and any combination thereof.
42 . (canceled)
43 . An S-RBD peptide immunogen construct having about 20 or more amino acids, represented by the formulae:
(Th) m -(A) n -(S-RBD B cell epitope peptide)-X
or
(S-RBD B cell epitope peptide)-(A) n -(Th) m -(Th) m -X
or
(Th) m -(A) n -(S-RBD B cell epitope peptide)-(A) n -(Th) m -X
wherein
Th is a heterologous T helper epitope;
A is a heterologous spacer;
(S1-RBD B cell epitope peptide) is a B cell epitope peptide having from 6 to about 35 amino acid residues from S1-RBD (SEQ ID NO: 226) or variants thereof;
X is an α-COOH or α-CONH2 of an amino acid;
m is from 1 to about 4; and
n is from 0 to about 10.
44 . The S1-RBD peptide immunogen construct according to claim 43 , wherein the S1-RBD B cell epitope peptide forms intra-disulfide bond to allow local constraint of the epitope selected from the group consisting of SEQ ID NOs: 23-24, 26-27, and 29-34.
45 . The S1-RBD peptide immunogen construct according to claim 43 , wherein the heterologous T helper is selected from the group consisting of SEQ ID NOs: 49-100.
46 . The S1-RBD peptide immunogen construct according to 43, wherein the S1-RBD B cell epitope peptide is selected from the group consisting of SEQ ID NOs: 23-24, 26-27, 29-34, and 315-319 and the Th epitope is selected from the group consisting of SEQ ID NOs: 49-100.
47 . The S1-RBD peptide immunogen construct according to 43, wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 107-144.
48 . An S1-RBD peptide immunogen construct comprising:
a. a B cell epitope comprising from about 6 to about 35 amino acid residues from the S1-RBD sequence of SEQ ID NO: 226;
b. a heterologous T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-100 and any combination thereof; and
c. an optional heterologous spacer selected from the group consisting of an amino acid, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 101), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 102), and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 103), and any combination thereof,
wherein the B cell epitope is covalently linked to the T helper epitope directly or through the optional heterologous spacer.
49 . The S1-RBD peptide immunogen construct according to claim 48 , wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 23-24, 26-27, 29-34, and 315-319.
50 . The S1-RBD peptide immunogen construct according to claim 48 , wherein the optional heterologous spacer is (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 101), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 102), or Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:103), where Xaa is any amino acid.
51 . The S1-RBD peptide immunogen construct according to claim 48 , wherein the T helper epitope is covalently linked to the amino- or carboxyl-terminus of the B cell epitope.
52 . The S1-RBD peptide immunogen construct according to claim 48 , wherein the T helper epitope is covalently linked to the amino- or carboxyl- of the B cell epitope through the optional heterologous spacer.
53 . A composition comprising the S1-RBD peptide immunogen construct according to claim 43 .
54 . A pharmaceutical composition comprising:
a. a peptide immunogen construct according to claim 43 ; and
b. a pharmaceutically acceptable delivery vehicle and/or adjuvant.
55 . The pharmaceutical composition according to claim 54 , wherein
a. the S1-RBD B cell epitope peptide is selected from the group consisting of SEQ ID NOs: 23-24, 26-27, 29-34, and 315-319;
b. the heterologous T helper epitope is selected from the group consisting of SEQ ID NOs: 49-100; and
c. the heterologous spacer is selected from the group consisting of an amino acid, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 101), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 102), and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 103), and any combination thereof; and
wherein the S1-RBD peptide immunogen construct is mixed with an CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.
56 . The pharmaceutical composition according to claim 54 , wherein
a. the S1-RBD peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 107-144; and
wherein the S1-RBD peptide immunogen construct is mixed with an CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.
57 . The pharmaceutical composition according to claim 56 , wherein the pharmaceutical composition further contains a separate peptide containing an endogenous SARS-CoV-2 Th epitope sequence of SEQ ID NOs: 13, 39-41, 44, 161-165, or any combination thereof.
58 . The pharmaceutical composition of 56 , wherein the pharmaceutical composition further contains a separate peptide containing an endogenous SARS-CoV-2 CTL epitope sequence of SEQ ID NOs: 9-12, 14-16, 19, 35-36, 42-43, 45-48, 145-160, or any combination thereof.
59 . The pharmaceutical composition according to claim 56 , wherein the pharmaceutical composition further contains
a. a separate peptide containing an endogenous SARS-CoV-2 Th epitope sequence of SEQ ID NOs: 13, 39-41, 44, 161-165, or any combination thereof; and
b. a separate peptide containing an endogenous SARS-CoV-2 CTL epitope sequence of SEQ ID NOs: 9-12, 14-16, 19, 35-36, 42-43, 45-48, 145-160, or any combination thereof.
60 . (canceled)
61 . (canceled)
62 . (canceled)
63 . (canceled)
64 . (canceled)
65 . (canceled)
66 . (canceled)
67 . A method of preventing and/or treating COVID-19 in an animal comprising administering the pharmaceutical composition according to claim 54 to the animal.
68 . A method of preventing and/or treating COVID-19 in an animal comprising administering the pharmaceutical composition of according to claim 57 to the animal.
69 . A method of preventing and/or treating COVID-19 in an animal comprising administering the pharmaceutical composition of according to claim 58 to the animal.
70 . A method of preventing and/or treating COVID-19 in an animal comprising administering the pharmaceutical composition of according to claim 59 to the animal.
71 . A fusion protein comprising:
a) an amino acid sequence derived from the extracellular domain (ECD) of human receptor ACE2 (ECD-hACE2) selected from the group consisting of SEQ ID NO: 228 and SEQ ID NO: 229
b) an optional hinge region from an IgG molecule selected from the group consisting of SEQ ID NO: 166-225; and
c) an Fc fragment of an IgG molecule selected from the group consisting of SEQ ID NOs: 231-234,
wherein the amino acid sequence in (a) is covalently linked to the Fc fragment in (c) through directly or through the optional hinge region in (b).
72 . The fusion protein according to claim 71 , wherein
the Fc fragment in (c) has an amino acid sequence of SEQ ID NO: 232 and
the optional hinge region has an amino acid sequence of SEQ ID NO: 166 or SEQ ID NO: 188.
73 . The fusion protein according to claim 71 , wherein the fusion protein is selected from the group consisting of: ACE2-ECD-sFc of SEQ ID NOs: 237, ACE2-ECDN-sFc of SEQ ID NO: 238, and ACE2-ECD-Fc of SEQ ID NO: 356.