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US-12616747-B2 - Stabilized beta-coronavirus antigens

US12616747B2US 12616747 B2US12616747 B2US 12616747B2US-12616747-B2

Abstract

Provided herein are engineered protein comprising stabilized coronavirus S protein ectodomains, such as stabilized SARS-CoV-2 S protein ectodomains. In some aspects, the engineered S protein ectodomains exhibit enhanced antigenicity. Methods are also provided for use of the engineered S protein ectodomains as diagnostics, in screening platforms, and/or in vaccine compositions.

Inventors

  • Jason McLellan
  • Ching-Lin Hsieh

Assignees

  • BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM

Dates

Publication Date
20260505
Application Date
20221202

Claims (20)

  1. 1 . An engineered protein comprising an engineered coronavirus S protein ectodomain that comprises a sequence at least 90% identical to: (a) positions 14-1208 of SEQ ID NO: 4; (b) positions 14-1160 of SEQ ID NO: 4; (c) positions 319-1208 of SEQ ID NO: 4; or (d) position 684-1205 of SEQ ID NO: 4, wherein the engineered protein comprises at least one pair of substitutions relative to the sequence of SEQ ID NO: 4 or 5, said at least one pair of substitutions being: Y707C/P792C, S704C/K790C, Q755C/N969C, G757C/S968C, G891C/P1069C, or S1030C/D1041C.
  2. 2 . The engineered protein of claim 1 , wherein the engineered protein comprises at least one pair of substitutions, said at least one pair of substitutions being: Y707C/P792C, S704C/K790C, A713C/L894C, Q755C/N969C, G757C/S968C, G891C/P1069C, S1030C/D1041C, or G1035C/V1040C.
  3. 3 . The engineered protein of claim 1 , wherein the engineered protein further comprises: (i) a substitution at a position corresponding to: T724, T752, T778, T961, I1013, H1058, S735, T859, I770, A1015, L727, S1021, Q901, S875, T912, H1088, L1141, V1040, L966, A766, T778, L938, V963, V911, N1108, V705, A893, N703, A672, A694, A1080, I1132, P862, T859, T547, N 978 , T961, S758, Q762, D1118, S659, S698, R1039, V722, A930, A903, Q913, S974, D979, P728, V951, V736, L858, S884, A893, P807, S875, T791, A879, G799, A924, V826, A899, Q779, F817, L865, T866, A892, A899, T912, A570, V963; T874, S1055, V729, A1022, L894, A713, L828, H1058, L822, A1056, Q965, S1003, A972, Q992, I980, A1078, V1133, H1088, T1120, I870, S1055, T1117, D1139, T1116, Y1138, I896, G885, Q901, F1103, P1112, G889, L1034, E819, S1055, A972, I980, I1081, N1135, E819, Q1054, Q957, I1130, V1040, H1088, V1104, R1000, A944, T724, A944, S730, S730, G769, A893, Q895, K921, L922, N978, A942, G946, S975, A890, S1003; and/or (ii) a deletion corresponding to positions 829-851, 675-686, 673-684, 1161-1208, or 1142-1208; and/or (iii) a substitution of two amino acids for amino acid positions 673-686.
  4. 4 . The engineered protein of claim 1 , comprising an engineered disulfide bond comprising paired cysteine substitutions at positions corresponding to: S735C and T859C; 1770C and A1015C; L727C and S1021C; V911C and N1108C; A672C and A694C; A1080C and I1132C; S659C and S698C; V722C and A930C; A903C and Q913C; S974C and D979C; P728C and V951C; V736C and L858C; S884C and A893C; P807C and S875C; T791C and A879C; G799C and A924C; A570C and V963C; T874C and S1055C; V729C and A1022C; L822C and A1056C; Q965C and S1003C; A972C and Q992C; I980C and Q992C; A1078C and V1133C; H1088C and T1120C; 1870C and S1055C; T1117C and D1139C; T1116C and Y1138C; I896C and Q901C; G885C and Q901C; F1103C and P1112C; G889C and L1034C; E819C and S1055C; A972C and I980C; I1081C and N1135C; or E819C and Q1054C.
  5. 5 . The engineered protein of claim 1 , comprising a cavity filling substitution selected from: T724M, I1013F, H1058W, Q901M, S875F, H1088W, L1141F, V1040F, T778L, L938F, V963L, R1039F, V826L, A899F, Q779M, L894F, H1058F, H1058Y, V1040Y, H1088Y, V1104I, R1000Y, R1000W, A944F, T724I, A944Y, S730L, A890V, D1118F, or S1003V.
  6. 6 . The engineered protein of claim 1 , comprising a proline substitution selected from: F817P, L865P, T866P, A892P, A899P, T912P, A893P, Q895P, K921P, L922P, N978P, A942P, G946P, or S975P.
  7. 7 . The engineered protein of claim 1 , comprising an electrostatic interaction substitution selected from: T752K, T912R, L828K, L828R, S730R, T961D, A766E, P862E, T859K, Q957E, G769E, T778Q, A713S, or I1130Y.
  8. 8 . The engineered protein of claim 1 , comprising a combination of at least one engineered disulfide bond, at least one cavity filling substitution, at least one proline substitution, and at least one electrostatic interaction substitution.
  9. 9 . The engineered protein of claim 1 , comprising the following substitutions relative to the sequence of SEQ ID NO: 4 or 5: F817P, A892P, A899P, A942P, K986P, and V987P.
  10. 10 . The engineered protein of claim 1 , comprising the following substitutions relative to SEQ ID NO: 4: F817P, A892P, A899P, A942P, K986P, V987P, and S704C/K790C.
  11. 11 . The engineered protein of claim 1 , comprising the following substitutions relative to SEQ ID NO: 4: F817P, A892P, A899P, A942P, K986P, V987P, S704C/K790C, and Q957E.
  12. 12 . The engineered protein of claim 1 , wherein the engineered coronavirus S protein ectodomain comprises a mutation that eliminates the furin cleavage site.
  13. 13 . The engineered protein of claim 1 , wherein the engineered protein does not comprise an S1 domain.
  14. 14 . The engineered protein of claim 1 , further comprising a stalk region positioned C-terminally relative to the ectodomain, wherein the stalk region has a sequence comprising or consisting of a sequence at least 95% identical to the sequence of amino acid 1143-1208 of SEQ ID NO: 4.
  15. 15 . The engineered protein of claim 1 , wherein the protein is fused or conjugated to a trimerization domain and/or a transmembrane domain.
  16. 16 . An engineered coronavirus trimer comprising at least one subunit comprising the engineered protein according to claim 1 .
  17. 17 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier; and the engineered protein of claim 1 .
  18. 18 . A nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence of the engineered protein of claim 1 .
  19. 19 . A method of preventing coronavirus infection or a disease associated with coronavirus infection in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 17 .
  20. 20 . A composition comprising the engineered protein of claim 1 bound to an antibody.

Description

REFERENCE TO RELATED APPLICATIONS The present application claims the priority benefit of U.S. provisional application No. 63/285,548, filed Dec. 3, 2021, the entire contents of which are incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant no. R01 AI127521 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on Nov. 30, 2022, is named UTSBP1294US_ST26.xml and is 50,214 bytes in size. BACKGROUND 1. Field The present disclosure relates generally to the fields of medicine, virology, immunology, and protein engineering. More particular, the disclosure relates to engineered protein comprising beta-coronavirus S protein ectodomains and the use thereof in drug design and vaccine formulation. 2. Description of Related Art An outbreak of COVID-19, the disease caused by infection of the coronavirus SARS-CoV-2, began in December 2019 in China has resulted in millions of infections and more than 100 thousand deaths. Like the virus that caused the SARS outbreak several years prior, SARS-CoV, the SARS-CoV-2 virus use their spike proteins to bind host cellular receptor angiotensin-converting enzyme 2 (ACE2). The interaction between the receptor binding domain (RBD) of the spike glycoprotein and the full-length human ACE2 protein. Although the sequence and structure of the SARS-CoV-2 spike protein is a known (see, e.g., Wrapp et al. 2020) there remains a need for stabilized S proteins that could be used for identifying drug candidates and for stimulating an effective immune response to the S protein. SUMMARY In one embodiment, provided herein are engineered proteins comprising an engineered coronavirus S protein ectodomain that comprises a sequence at least 90% identical to: (a) positions 14-1208 of SEQ ID NO: 4; (b) positions 14-1160 of SEQ ID NO: 4; (c) positions 319-1208 of SEQ ID NO: 4; or (d) position 684-1205 of SEQ ID NO: 4, wherein the engineered protein comprises at least one substitution relative to the sequence of SEQ ID NO: 4 or 5, said at least one substitution being: P792C, S704C, K790C, A713C, L894C, Q755C, N969C, G757C, S968C, G891C, P1069C, S1030C, D1041C, G1035C, or V1040C. In some aspects, the engineered protein comprises at least one pair of substitutions, said at least one pair of substitutions being: Y707C/P792C, S704C/K790C, A713C/L894C, Q755C/N969C, G757C/S968C, G891C/P1069C, S1030C/D1041C, or G1035C/V1040C. In some aspects, the engineered protein further comprises a substitution of Y707C and/or T883C. In some aspects, the engineered protein further comprises a pair of substitutions, said at least one pair of substitutions being: Y707C/T883C. In some aspects, the engineered protein further comprises an additional engineered disulfide bond, a cavity filling substitution, and/or a substitution that provides an electrostatic or polar interaction. In some aspects, the engineered protein further comprises: (i) a substitution at a position corresponding to: T724, T752, T778, T961, I1013, H1058, S735, T859, I770, A1015, L727, S1021, Q901, S875, T912, H1088, L1141, V1040, L966, A766, T778, L938, V963, V911, N1108, V705, A893, N703, A672, A694, A1080, I1132, P862, T859, T547, N978, T961, S758, Q762, D1118, S659, S698, R1039, V722, A930, A903, Q913, S974, D979, P728, V951, V736, L858, S884, A893, P807, S875, T791, A879, G799, A924, V826, A899, Q779, F817, L865, T866, A892, A899, T912, A570, V963; T874, S1055, V729, A1022, L894, A713, L828, H1058, L822, A1056, Q965, S1003, A972, Q992, I980, A1078, V1133, H1088, T1120, I870, S1055, T1117, D1139, T1116, Y1138, I896, G885, Q901, F1103, P1112, G889, L1034, E819, S1055, A972, I980, I1081, N1135, E819, Q1054, Q957, I1130, V1040, H1088, V1104, R1000, A944, T724, A944, S730, S730, G769, A893, Q895, K921, L922, N978, A942, G946, S975, A890, S1003; and/or (ii) a deletion corresponding to positions 829-851, 675-686, 673-684, 1161-1208, or 1142-1208; and/or (iii) a substitution of two amino acids for amino acid positions 673-686. In some aspects, the engineered protein comprises an engineered disulfide bond comprising paired cysteine substitutions at positions corresponding to: S735C and T859C; I770C and A1015C; L727C and S1021C; V911C and N1108C; A672C and A694C; A1080C and I1132C; S659C and S698C; V722C and A930C; A903C and Q913C; S974C and D979C; P728C and V951C; V736C and L858C; S884C and A893C; P807C and S875C; T791C and A879C; G799C and A924C; A570C and V963C; T874C and S1055C; V729C and A1022C; L822C and A1056C; Q965C and 51003C; A972C and Q992C; I980C and Q992C; A1078C and V1133C; H1088C and T1120C; I870C and S1055C; T1117C and D1139C; T1116C and Y1138C; I896C and Q901C; G885C and Q901C; F1103C and P1112C; G889C and L1034