BACKGROUND OF THE INVENTION

Filoviruses such as Zaire Ebola virus (EBOV) and Marburg virus can cause a lethal hemorrhagic fever in humans and nonhuman primates (NHPs). The virus glycoprotein, GP, mediates cell entry by initiating attachment and membrane fusion. Structures of GP-bound neutralizing antibodies (NAbs) from human survivors demonstrated that GP harbors all NAb epitopes known to date and is the sole target for vaccine design. Like HIV-1 envelope glycoprotein (Env), filovirus GP is metastable and produces non-functional forms when expressed in various cell lines.

A lasting campaign has been undertaken to identify and characterize NAbs for EBOV and other filoviruses such as Marburg virus (MARV). As a result, large panels of NAbs were isolated from human survivors, vaccinated humans, and immunized animals. Crystallography and electron microscopy (EM) have revealed multiple sites of vulnerability on EBOV GP. A recent study of 171 monoclonal antibodies (mAbs) defined eight epitope classes, of which six can be recognized by broadly neutralizing antibodies (bNAbs). While recombinant virus-like particles (VLPs) can protect against EBOV challenge in animals, the difficulties in manufacturing have hampered their further development as human vaccines.

Despite recent advances in the development of medical regimen against filoviruses, there is still a strong need in the art for more effective means for treating and preventing human infections and deaths caused by Ebolaviruses. The instant invention is directed to this and other unmet needs in the art.

SUMMARY OF THE INVENTION

In one aspect, the invention provides engineered or redesigned Ebolavirus glycoprotein (GP) sequences. Relative to a wildtype Ebolavirus GP sequence, the engineered sequences contain (a) a substitution of residue W615 in heptad repeat 2 (HR2) with a smaller hydrophobic residue, and (b) one or more proline substitutions in heptad repeat 1 C segment (HR1C). The amino acid numbering is based on Zaire Ebolavirus GP sequence that has the ectodomain sequence identified by UniProt ID Q05320 (SEQ ID NO:1). In various embodiments, the engineered Ebolavirus GP sequences can be modified or derived from the wildtype GP sequence of a Zaire Ebolavirus (EBOV), a Sudan virus (SUDV), a Taï forest virus (TAFV), a Bundibugyo virus (BDBV), or a Reston Ebolavirus (RESTV).

In some embodiments, residue W615 in the engineered Ebolavirus GP sequences can be replaced with residue L, A, V, I or F. In some embodiments, the proline substitutions in HR1Cinclude T577P and/or L579P. In some embodiments, the engineered Ebolavirus GP sequences additionally include a truncation at the C-terminus of the membrane proximal external region (MPER). In some of these embodiments, the engineered Ebolavirus GP sequences can further include (a) a deletion of the mucin-like domain (MLD) deleted from the GP1 subunit, and/or (b) a deletion of MPER from the GP2 subunit.

In another aspect, the invention provides engineered Ebolavirus GP sequences that contain (a) a truncated soluble GP of an Ebolavirus that has MLD deleted from the GP1 subunit and MPER deleted from the GP2 subunit, and (b) one or more modifications in the HR2 and HR1 regions. In various embodiments, the additional modifications in the HR2 and HR1 regions can be (i) substitution of W615 in heptad repeat 2 (HR2) with a smaller hydrophobic residue, (ii) an extension of HR2 at the C terminus, (iii) one or more proline substitutions in heptad repeat 1 C segment (HR1C), and (iv) one or more engineered inter-GP disulfide bonds. Again, the amino acid numbering is based on the Zaire Ebolavirus GP sequence with UniProt ID Q05320 (SEQ ID NO:1). In various embodiments, these engineered Ebolavirus GP sequences can be derived from Zaire Ebolaviruses (EBOV), Sudan viruses (SUDV), Taï forest viruses (TAFV), Bundibugyo viruses (BDBV), or Reston Ebolaviruses (RESTV). Some engineered Ebolavirus GP sequences are based on the truncated soluble GP sequence shown in SEQ ID NO:2 or SEQ ID NO:3, or a conservatively modified or substantially identical variant thereof. In some embodiments, the engineered GP sequences can have a N-terminal leader sequence. As exemplification, the N-terminal leader sequence can contain SEQ ID NO:41 or a conservatively modified variant.

Some engineered soluble Ebolavirus GP sequences contain a substitution of residue W615. In some of these embodiments, residue W615 can be replaced with L, A, V, I or F. Some of these engineered Ebolavirus GP proteins contain the amino acid sequence shown in SEQ ID NOs:4, a conservatively modified variant or a substantially identical sequence thereof. In some embodiments, the engineered soluble Ebolavirus GP proteins can contain an extension of HR2 at the C-terminus. In some of these embodiments, the HR2 extension can be (a) extending HR2 C terminus with a N-terminal fragment of adjacent MPER of the Ebolavirus glycoprotein or (b) replacing a HR2 C-terminal fragment with a longer leucine zipper motif. As exemplification, HR2 extension in the engineered soluble Ebolavirus GP proteins can include (a) extension of the HR2 C terminus from residue 632 to residue 637 in MPER, i.e., addition of residues KTLPD (SEQID NO:32), (b) extension of the HR2 C terminus from residue 632 to residues 643 in MPER, i.e., addition of residues KTLPDQGDNDN (SEQ ID NO:33), or (c) replacement of residues 617-632 with a GCN4 leucine zipper sequence shown in SEQ ID NO:34.

Some engineered soluble Ebolavirus GP sequences of the invention contain both a HR2 extension noted above and a W615 substitution in HR2. In some of these embodiments, the W615 substitution can be W615L or P612G/W615F double mutation. In some embodiments, the engineered GP sequences contain (a) W615L substitution and (b) extension of the HR2 C terminus from residue 632 to residue 637 in MPER of the Ebolavirus GP. In some other embodiments, the engineered GP sequences contain (a) P612G/W615F double mutation in HR2 and (b) replacement of residues 617-632 with a GCN4 leucine zipper sequence shown in SEQ ID NO:34. Some specific engineered soluble Ebolavirus GP sequences contain an amino acid sequence shown in any one of SEQ ID NOs:5-8, a conservatively modified variant or a substantially identical sequence thereof.

In addition to the HR2 extension and/or the W612 substitution, the engineered GP sequences in some embodiments can also contain a C-terminal trimerization motif. In some of these embodiments, the C-terminal trimerization motif can contain SEQ ID NO:29 or SEQ ID NO:30, or a conservatively modified variant or a substantially identical sequence thereof. In addition to the W612 substitution, the engineered GP sequences in some embodiments can also contain one or more proline substitutions in the HR1Csegment. Any residue in HR1Ccan be replaced with a proline residue. In some of these embodiments, the proline substitution comprises T577P or L579P. Some specific examples of engineered Ebolavirus GP sequences having a W612 substitution in HR2 and a proline substitution in HR1Ccontain an amino acid sequence as set forth in any one of SEQ ID NOs:9-16, a conservatively modified variant or a substantially identical sequence thereof.

Some engineered GP sequences of the invention contain a HR2 extension, a proline substitution in HR1C, and a substitution at residue W615. In some of these embodiments, the W615 residue can be replaced with L, A, V, I or F. In some of the embodiments, the engineered Ebolavirus GP sequences contain an extension of HR2 C terminus from residue 632 to residue 637 in MPER. Some specific examples of such engineered Ebolavirus GP sequences contain an amino acid sequence as set forth in any one of SEQ ID NOs:17 and 20, a conservatively modified variant or a substantially identical sequence thereof. In some of the embodiments, the engineered Ebolavirus GP sequences can further include a C-terminal trimerization motif. For example, the engineered Ebolavirus GP proteins can include the C-terminal trimerization motif shown in SEQ ID NO:29 or SEQ ID NO:30, or a conservatively modified variant or a substantially identical sequence thereof.

Some engineered Ebolavirus GP sequences contain (a) W615L substitution, (b) proline substitution T577P or L579P, and (c) HR2 extension from residue 632 to residue 637 in MPER. Some specific examples of these engineered Ebolavirus GP sequences contain amino acid sequence as set forth in any one of SEQ ID NOs:18, 19, 21 and 22, a conservatively modified variant or a substantially identical sequence thereof.

Some engineered Ebolavirus GP sequences contain one or more engineered disulfide bonds between two neighboring GP protomers. In various embodiments, the engineered disulfide bonds scan be created by cysteine substitutions at residue pairs G91/A575 (SS2), F153/Y534 (SS1), T520/A575 (SS3), G157/I532 (SS4), D522/A575 (SS5), and/or K56/G599 (SS6). Some specific examples of these engineered Ebolavirus GP sequences have an amino acid sequence are shown in any one of SEQ ID NOs:23-28, a conservatively modified variant or a substantially identical sequence thereof.

In some related aspects, the invention provides nucleic acid or polynucleotide sequences that encode an engineered full length or truncated Ebolavirus GP protein sequence described herein. Some engineered Ebolavirus GP sequences also contain a leader peptide encoding sequence at the 5′. Also provided in the invention are pharmaceutical compositions that contain an engineered Ebolavirus GP protein or encoding polynucleotide sequence described herein, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides vaccine compositions that contain an engineered Ebolavirus GP protein that is displayed on the surface of a self-assembling nanoparticle. In some embodiments, C-terminus of the engineered Ebolavirus GP protein is fused to the N-terminus of subunit of the self-assembling nanoparticle (NP). In some embodiments, C-terminus of the engineered Ebolavirus GP protein is fused to the subunit of the self-assembling nanoparticle via a linker sequence GGGGS (SEQ ID NO=76,r (GGGGS)2(SEQ ID NO:42). In some embodiments, the engineered Ebolavirus GP proteins in the NP vaccine compositions contain an amino acid sequence as set forth in any one of SEQ ID NOs:4-28, a conservatively modified variant or a substantially identical sequence thereof. In some embodiments, subunit of the self-assembling nanoparticle contains the polypeptide sequence as shown in SEQ ID NO:36 (E2p), SEQ ID NO:37 (I3-01v9), or SEQ ID NO:38 (ferritin), a conservatively modified variant or a substantially identical sequence thereof.

Some NP scaffolded vaccine compositions of the invention can additionally contain a locking domain (LD) that is fused to the C terminus of the NP subunit sequence. In some of these embodiments, the employed LD contains the sequence shown in SEQ ID NO:39 or 40, a conservatively modified variant or a substantially identical sequence thereof. Some of the scaffolded vaccines can additionally contain a T-cell epitope that is fused to the C-terminus of the locking domain. In some of these embodiments, the employed T-cell epitope contains the sequence shown in SEQ ID NO:31, a conservatively modified variant or a substantially identical sequence thereof.

Some NP vaccine compositions of the invention contain (1) a polypeptide sequence containing from N terminus to C terminus (a) an engineered Ebolavirus GP protein, linker sequence G4S (SEQ ID NO:43), nanoparticle sequence ferritin, (b) an engineered Ebolavirus GP protein, linker sequence G4S (SEQ ID NO:43), nanoparticle sequence E2p, or (c) an engineered Ebolavirus GP protein, linker sequence (G4S)2(SEQ ID NO:42), nanoparticle sequence I3-01v9; or (2) a conservatively modified variant of the polypeptide sequence of (1). In some of these embodiments, the engineered Ebolavirus GP sequences contain W615L substitution, proline substitution T577P, and HR2 extension from residue 632 to residue 637 in MPER. These engineered Ebolavirus GP sequences can additionally contain a locking domain and/or a T-cell epitope that is fused to the C-terminus of the nanoparticle subunit sequence. In some embodiments, the vaccine compositions contain (1) a polypeptide sequence containing from N-terminus to C-terminus (a) the engineered Ebolavirus GP protein shown in SEQ ID NO:17, nanoparticle subunit sequence shown in SEQ ID NO:36 (E2p), locking domain shown in SEQ ID NO:39 (LD4), and T cell epitope shown in SEQ ID NO:31 (PADRE) or (b) the engineered Ebolavirus GP protein shown in SEQ ID NO:17, nanoparticle sequence shown in SEQ ID NO:37 (I3-01v9), locking domain shown in SEQ ID NO:40 (LD7), or (2) a conservatively modified variant of the polypeptide sequence.

A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

