Granted Patent
B2
US 12,344,635 · App. 17/211,379 · Granted Jul 1, 2025
COVID-19 vaccine
Inventors:
Joseph T. Barbieri (New Berlin, WI); Amanda Przedpelski (Milwaukee, WI); Eric A. Johnson (Madison, WI); Sabine Pellett (Madison, WI); William H. Tepp (Stoughton, WI)
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Abstract
Provided herein are antigenic peptides comprising the SARS-COV-2 spike protein receptor binding domain (CRBD) polypeptide or portions thereof, linked to a non-catalytic, non-toxic tetanus toxin variant (i.e., a modified tetanus toxin or “MTT”) and vaccine compositions comprising the same. In addition, provided herein are methods for making and using CRBD-MTT fusion proteins as immunogenic agents.
Claims (30)
1. A fusion protein comprising
(i) a modified tetanus toxin (MTT) polypeptide comprising a sequence having at least 95% identity to SEQ ID NO:1, having a mutation at each of positions R372 and Y375, and having a mutation at two or more positions selected from E234, K768, R1226, and W1289, wherein each position is numbered relative to SEQ ID NO:1, the MTT polypeptide having reduced toxicity and receptor binding compared to the toxicity and receptor binding of SEQ ID NO: 1; and
(ii) a SARS-COV-2spike protein receptor binding domain (CRBD) polypeptide or a portion thereof.
2. The fusion protein of claim 1 , wherein the CRBD polypeptide or a portion thereof comprises at least a portion of SEQ ID NO:3.
3. The fusion protein of claim 1 , wherein the CRBD polypeptide or a portion thereof comprises a sequence having at least 95% identity to SEQ ID NO:3.
4. The fusion protein of claim 1 , wherein the CRBD polypeptide or a portion thereof comprises an amino acid sequence corresponding to residues 376-525 or 433-524 of SEQ ID NO: 2.
5. The fusion protein of claim 1 , wherein the MTT polypeptide and the CRBD polypeptide are connected by a linker polypeptide.
6. The fusion protein of claim 5 , wherein the linker polypeptide is a poly-glycine sequence.
7. The fusion protein of claim 1 , wherein the MTT polypeptide and the CRBD polypeptide are chemically cross-linked.
8. The fusion protein of claim 1 , wherein in the MTT polypeptide amino acid R at position R372 is replaced with amino acid A, and wherein amino acid Y at position Y375 is replaced with amino acid F.
9. The fusion protein of claim 1 , wherein the MTT polypeptide mutations comprise R372A, Y375F, E234Q, R1226L, and W1289A.
10. The fusion protein of claim 9 , wherein the MTT polypeptide comprises SEQ ID NO:5.
11. The fusion protein of claim 1 , wherein the MTT polypeptide mutations comprise R372A, Y375F, E234Q, K768A, R1226L, and W1289A.
12. The fusion protein of claim 11 , wherein the MTT polypeptide comprises SEQ ID NO:6.
13. The fusion protein of claim 1 , wherein the MTT polypeptide further comprises a mutation at one or both of positions L230 and Y26, wherein each position is numbered relative to SEQ ID NO: 1.
14. The fusion protein of claim 13 , wherein the mutations at one or both of positions L230 and Y26 comprise L230K and Y26A.
15. The fusion protein of claim 14 , wherein the MTT polypeptide comprises SEQ ID NO:7 or SEQ ID NO:8.
16. The fusion protein of claim 1 , wherein the fusion protein comprises a sequence at least 95% identical to SEQ ID NO:10.
17. A polynucleotide encoding the fusion protein of claim 1 .
18. The polynucleotide of claim 17 , wherein the polynucleotide comprises a sequence at least 95% identical to SEQ ID NO:9.
19. A vector comprising the polynucleotide of claim 18 .
20. A composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier.
21. A method of reducing the risk of a subject developing COVID-19 by inducing an immune response through administering to the subject a therapeutically effective amount of the fusion protein of claim 1 .
22. A method for producing a CRBD-MTT chemically cross-linked fusion protein comprising:
obtaining a polypeptide composition comprising (i) a modified tetanus toxin (MTT) polypeptide comprising a sequence having at least 95% identity to SEQ ID NO:1, having a mutation at each of positions R372 and Y375, and having a mutation at two or more positions selected from E234, K768, R1226, and W1289, wherein each position is numbered relative to SEQ ID NO:1, and (ii) a SARS-COV-2 spike protein receptor binding domain (CRBD) polypeptide or a portion thereof; and
contacting the polypeptide composition with a crosslinking agent for a time and under conditions sufficient to chemically crosslink the CRBD polypeptide and the MTT polypeptide; whereby a CRBD-MTT chemically cross-linked fusion protein is produced.
23. The method of claim 22 , wherein the crosslinking agent is selected from the group consisting of formaldehyde, disuccinimidyl suberate (DSS), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), ethylene glycol bis(sulfosuccinimidylsuccinate) (Sulfo-EGS), bis(sulfosuccinimidyl)suberate (BS3), and dithiobis(succinimidylpropionate) (DSP).
24. The method of claim 22 , wherein the CRBD polypeptide comprises a sequence having at least 95% identity to SEQ ID NO:3.
25. The method of claim 22 , wherein the CRBD polypeptide or a portion thereof comprises an amino acid sequence corresponding to residues 433-524 of SEQ ID NO:2.
26. The method of claim 22 , wherein the CRBD polypeptide or a portion thereof comprises an amino acid sequence corresponding to residues 376-525 of SEQ ID NO:2.
Assignments (3)
CONFIRMATORY LICENSE
Recorded Nov 3, 2023
From: MEDICAL COLLEGE OF WISCONSIN
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 065456/0105 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Mar 29, 2021
From: JOHNSON, ERIC; PELLETT, SABINE; TEPP, WILLIAM
To: WISCONSIN ALUMNI RESEARCH FOUNDATION
Reel/Frame 055753/0428 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Mar 26, 2021
From: BARBIERI, JOSEPH T.; PRZEDPELSKI, AMANDA
To: THE MEDICAL COLLEGE OF WISCONSIN, INC.
Reel/Frame 055736/0712 →
Continuity (3)
Provisional Application
63104360
· Oct 22, 2020
Provisional Application
62994081
· Mar 24, 2020
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