Human cytomegalovirus GB polypeptide

Patent metadata

Field	Value
Publication number	US-11857622-B2
Application number	US-202117350008-A
Country	US
Kind code	B2
Filing date	Jun 17, 2021
Priority date	Jun 21, 2020
Publication date	Jan 2, 2024
Grant date	Jan 2, 2024

Abstract

The present invention comprises mutants of a wild-type cytomegalovirus (CMV) glycoprotein B (gB) protein that include at least two introduced amino acid mutations relative to the amino acid sequence of the wild-type HCMV glycoprotein B (gB), compositions including the mutants and uses thereof. In some embodiments, the mutant is stabilized in a conformation alternative to the gB postfusion conformation.

First claim

The invention claimed is:

1. An engineered mutant of a wild-type cytomegalovirus (CMV) glycoprotein B (gB) protein, wherein said mutant comprises at least two amino acid mutations, and wherein said at least two amino acid mutations comprise at least one engineered disulfide mutation selected from the group consisting of: D217C and Y589C; M371C and W506C; and N524C and M684C; and wherein the numbering of the engineered mutations is relative to SEQ ID NO:1 numbering.

2. The mutant according to claim 1 , wherein the amino acid mutations comprise a combination of at least two engineered disulfide mutations and at least one additional mutation.

3. The mutant according to claim 1 , which is in the form of a trimer.

4. The mutant according to claim 1 , which has increased stability in prefusion form as compared with the wild-type CMV gB protein, wherein the stability is measured by binding of a prefusion-specific antibody, thermal shift assay or EM imaging.

5. The mutant according to claim 1 , wherein the wild-type CMV gB sequence is from the Towne strain.

6. The mutant according to claim 1 , wherein said gB comprises an additional mutation selected from the group consisting of: (1) substitution of YIH at positions 155-157 with GHR; (2) substitution of W at position 240 with A; (3) substitution of C at position 246 with S; (4) substitution of P at position 655 with S; (5) substitution of F at position 678 with S; (6) substitution of L at position 680 with T; (7) substitution of R at position 685 with A; (8) substitution of MIALDI at positions 648-653 with GSGKDG; (9) substitution of R at position 693 with V; (10) substitution of I at position 675 with S; (11) substitution of I at positions 767 and 768 with C; (12) substitution of D at position 703 and P at position 704 with C; and (13) substitution of Y at position 696 and V at position 697 with C.

7. The mutant according to claim 1 , wherein said gB comprises at least one additional mutation, and wherein the additional mutation is selected from the group consisting of: (1) substitution of YIH at positions 155-157 with GHR; (2) substitution of W at position 240 with A; (3) substitution of C at position 246 with S; (4) substitution of P at position 655 with S; (5) substitution of F at position 678 with S; (6) substitution of L at position 680 with T; (7) substitution of R at position 685 with A; (8) substitution of MIALDI at positions 648-653 with GSGKDG; (9) substitution of R at position 693 with V; (10) substitution of I at position 675 with S; (11) substitution of I at positions 767 and 768 with C; (12) substitution of D at position 703 and P at position 704 with C; and (13) substitution of Y at position 696 and V at position 697 with C.

8. The mutant according to claim 1 , wherein the amino acid sequence of the mutant does not comprise a signal sequence (residues 1-22 of SEQ ID NO: 1).

9. The mutant of claim 1 , wherein the amino acid sequence of the mutant does not comprise a membrane-proximal region (MPR) (residues 705-750 of SEQ ID NO: 1), transmembrane (TM) domain (residues 751-772 of SEQ ID NO: 1) or cytoplasmic (CT) domain domain (residues 773-907 of SEQ ID NO: 1).

10. The mutant of claim 1 , wherein the amino acid sequence of the mutant comprises a truncated Domain V region.

11. The mutant of claim 1 , wherein the amino acid sequence of the mutant does not comprise a Domain V region (residues 649-707 of SEQ ID NO: 1).

12. The mutant of claim 1 , wherein the mutant further comprises a trimerization motif linked to the C terminus of the mutant.

13. The mutant of claim 12 , wherein the trimerization motif is selected from the group consisting of:
(i) an inter-protomer disulfide ring;
(ii) GCN4;
(iii) T4 fibritin foldon; and
(iv)C-terminus fusion sequence.

14. The mutant of claim 1 , wherein the wildtype CMV gB polypeptide sequence is selected from SEQ ID NOs: 1, 107-140, or 224.

15. The mutant of claim 1 , wherein the wildtype CMV gB polypeptide sequence is encoded by the polynucleotide sequences set forth in SEQ ID NOs: 225-254.

16. A nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence of a CMV gB protein mutant according to claim 1 .

17. A pharmaceutical composition comprising (i) a CMV gB protein mutant according to claim 1 and (ii) a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17 , which is a vaccine.

19. A method of reducing CMV infection in a subject comprising administering to the subject an effective amount of the vaccine according to claim 18 .

20. A method of eliciting an immune response to CMV infection in a subject comprising administering to the subject an effective amount of the vaccine according to claim 18 .

21. A method of inhibiting CMV infection in a subject comprising administering to the subject an effective amount of the vaccine according to claim 18 .

22. The mutant according to claim 7 , wherein said gB comprises an additional mutation selected from the group consisting of: (1) substitution of YIH at positions 155-157 with GHR; (2) substitution of W at position 240 with A; (3) substitution of C at position 246 with S; and (4) substitution of I at position 675 with S.

23. The mutant according to claim 7 , wherein the amino acid mutations are a combination of mutations selected from the group consisting of:
(1) combination of D217C and Y589C, M371C and W506C, and 1675S;
(2) combination of D217C and Y589C, N524C and M684C, and 1675S;
(3) combination of D217C and Y589C, M371C and W506C, Y155G 1156H H157R, W240A, C246S and 1675S; and
(4) combination of D217C and Y589C, N524C and M684C, Y155G 1156H H157R, W240A, C246S and 1675S.

24. The mutant according to claim 1 , wherein the mutant comprises a cysteine (C) at position 217 (217C) and at position 589 (589C), a cysteine (C) at position 371 (371C) and at position 506 (506C), and a serine (S) at position 675 (675S), and wherein the mutant is selected from the group consisting of:
(1) a mutant comprising the amino acid sequence set forth in SEQ ID NO:257; and
(2) a mutant comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:257.

25. The mutant according to claim 1 , wherein the mutant comprises a cysteine (C) at position 217 (217C) and at position 589 (589C), a cysteine (C) at position 524 (524C) and at position 684 (684C), and a serine (S) at position 675 (675S), and wherein the mutant is selected from the group consisting of:
(1) a mutant comprising the amino acid sequence of SEQ ID NO: 259; and
(2) a mutant comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 259.

26. The mutant according to claim 1 , wherein the mutant comprises a cysteine (C) at position 217 (217C) and at position 589 (589C), a cysteine (C) at position 371 (371C) and at position 506 (506C), a serine (S) at position 675 (675S), a glycine (G) at position 155 (155G), a histidine at position 156 (156H), an arginine at position 157 (157R), an alanine at position 240 (240A), and a serine at position 246 (246S), and wherein the mutant is selected from the group consisting of:
(1) a mutant comprising the amino acid sequence set forth in SEQ ID NO:261; and
(2) a mutant comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 261.

Assignees

• Pfizer

Inventors

• Liu Yuhang
• Che Ye
• Chi Xiaoyuan Sherry
• Dormitzer Philip Ralph
• Nicki Jennifer Anne
• Yao Xiaojie

Classifications

• A61K39/245 Primary

  Herpetoviridae, e.g. herpes simplex virus · CPC title

• C07K14/045

  Cytomegalovirus · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N2710/16134

  Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein · CPC title

• A61K39/12 Primary

  Viral antigens · CPC title