Modified cytomegalovirus proteins and stabilized complexes
Described are mutant human cytomegalovirus (HCMV) pentamer complex polypeptides, methods of making them, and their use in HCMV protein complexes and compositions. In particular, the use of the modified HCMV polypeptides to stabilize HCMV complexes or unmask a pentamer epitope is described.
1. A complex comprising one or more mutant Human Cytomegalovirus (HCMV) polypeptides, wherein the one or more mutant HCMV polypeptides are:
(a) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine (C) at residue 142, numbered with respect to SEQ ID NO: 13;
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cysteine (C) at residue 150, numbered with respect to SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 64, numbered with respect to SEQ ID NO: 17, and a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;
(b) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine (C) at residue 142, numbered with respect to SEQ ID NO: 13;
a gL polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 150, numbered with respect to SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 64, numbered with respect to SEQ ID NO: 17, and a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;
(c) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine (C) at residue 142, numbered with respect to SEQ ID NO: 13;
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 145, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cysteine (C) at residue 150, numbered with respect to SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 64, numbered with respect to SEQ ID NO: 17, and a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;
(d) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine
(C) at residue 142, numbered with respect to SEQ ID NO: 13;
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 224, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cysteine (C) at residue 150, numbered with respect to SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 64, numbered with respect to SEQ ID NO: 17, and a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;
(e) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine
(C) at residue 142, numbered with respect to SEQ ID NO: 13;
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 224, numbered with respect to the wild-type Sequence of SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;
(f) a pUL128 polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 77, numbered with respect to the wild-type Sequence of SEQ ID NO: 13, and has a cavity filling mutant at residue 103, numbered with respect to the wild-type Sequence of SEQ ID NO: 13; and
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cavity filling mutant at residue 145, numbered with respect to the wild-type Sequence of SEQ ID NO: 7;
(g) a pUL128 polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 77, numbered with respect to the wild-type Sequence of SEQ ID NO: 13, and has a cavity filling mutant at residue 103, numbered with respect to the wild-type Sequence of SEQ ID NO: 13; and
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, has a cavity filling mutant at residue 145, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cavity filling mutant at residue 224, numbered with respect to the wild-type Sequence of SEQ ID NO: 7;
(h) a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7; and
a pUL131 polypeptide, or complex-forming fragment thereof, that has a repacking hydrophobic mutant at residue 52, numbered with respect to the wild-type Sequence of SEQ ID NO: 21, and has a repacking hydrophobic mutant at residue 67, numbered with respect to the wild-type Sequence of SEQ ID NO: 21;
(i) a pUL131 polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 86, numbered with respect to the wild-type Sequence of SEQ ID NO: 21; or
(j) a pUL128 polypeptide, or complex-forming fragment thereof, that has a cysteine
(C) at residue 83, numbered with respect to SEQ ID NO: 13; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine
(C) at residue 167, numbered with respect to SEQ ID NO: 17.
2. The complex of claim 1 , wherein the cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), isoleucine (I), or leucine (L).
3. The complex of claim 1 , wherein the cavity filling mutant at residue 145, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), isoleucine (I), or leucine (L).
4. The complex of claim 1 , wherein the cavity filling mutant at residue 224, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), isoleucine (I), or leucine (L).
5. The complex of claim 1 , wherein the cavity filling mutant at residue 77, numbered with respect to the wild-type Sequence of SEQ ID NO: 13, is tryptophan (W), phenylalanine (F), tyrosine (Y), isoleucine (I), or leucine (L).
6. The complex of claim 1 , wherein the cavity filling mutant at residue 103, numbered with respect to the wild-type Sequence of SEQ ID NO: 13, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), or isoleucine (I).
7. The complex of claim 1 , wherein the repacking hydrophobic mutant at residue 52, numbered with respect to the wild-type Sequence of SEQ ID NO: 21, is tryptophan (W), phenylalanine (F), methionine (M), cysteine (C), alanine (A), leucine (L), isoleucine (I), valine (V) or proline (P).
8. The complex of claim 1 , wherein the repacking hydrophobic mutant at residue 67, numbered with respect to the wild-type Sequence of SEQ ID NO: 21, is tryptophan (W), phenylalanine (F), methionine (M), cysteine (C), leucine (L), isoleucine (I), valine (V) or proline (P).
9. The complex of claim 1 , wherein the cavity filling mutant at residue 86, numbered with respect to the wild-type Sequence of SEQ ID NO: 21, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), isoleucine (I), or leucine (L).
10. The complex of claim 1 , wherein the complex has an increased thermostability as compared to a control complex in the same conditions.
11. The complex of claim 1 , wherein the complex is an HCMV pentamer complex comprising one or more mutant HCMV polypeptides.
12. An immunogenic composition comprising the complex of claim 1 .
13. An isolated nucleic acid molecule comprising one or more operably linked polynucleotide sequences that encode the complex of claim 1 .
14. An expression vector comprising the isolated nucleic acid molecule of claim 13 .
15. An isolated host cell comprising the isolated nucleic acid molecule of claim 13 .
16. An isolated host cell comprising the expression vector of claim 14 .
17. A method of inducing an immune response against Human Cytomegalovirus (HCMV), comprising administering to a subject an immunologically effective amount of the immunogenic composition of claim 12 .
18. A method of inhibiting Human Cytomegalovirus (HCMV) entry into a cell, comprising contacting the cell with a complex according to claim 1 .