HBV polymerase mutants
Inventors
Martin, Perrine • Silvestre, Nathalie • Marchand, Jean-Baptiste
Assignees
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Publication Number
US-10190105-B2
Publication Date
2019-01-29
Expiration Date
Abstract
The present invention relates to polymerase HBV mutant polypeptides comprising a mutated polymerase domain which is functionally disrupted for polymerase activity and fusion proteins comprising such polymerase mutant polypeptide. The present invention also relates to a nucleic acid molecule and an expression vector for expressing said polymerase mutant polypeptide as well as a composition which can be used for eliciting an immune response to HBV with the goal of providing a protective or therapeutic effect against HBV infection.
Core Innovation
The invention relates to mutant polypeptides comprising a mutated HBV polymerase domain with an internal deletion that functionally disrupts polymerase activity. The internal deletion comprises at least 4 amino acid residues and at most 30 amino acid residues, and the mutated polymerase domain comprises the amino acid sequence shown in SEQ ID NO:1 but lacks at least the Tyr residue in position 203, the Met residue in position 204, the Asp residue in position 205 and the Asp residue in position 206, the Val residue in position 207, the Val residue in position 208, and the Leu residue in position 209.
In further embodiments, the mutant polypeptide comprises a mutated RNaseH domain that functionally disrupts RNaseH activity of a native HBV polymerase. The disclosure includes deletions and substitutions based on defined mutation options, including deletions of at least 8 amino acids and at most 60 amino acids within a defined segment and specified residue substitutions at defined positions.
The invention further includes fusion proteins combining the mutant polypeptide with HBV core polypeptides and, in certain embodiments, incorporating one or two HBsAg immunogenic domains. In these fusion proteins, the core polypeptide may be N-terminal to the mutant polymerase-derived component, and the HBsAg immunogenic domains replace the internal deletions in the mutated polymerase domain and/or the deletion in the mutated RNaseH domain.
The disclosure additionally covers nucleic acids, vectors, host cells, and pharmaceutical compositions for eliciting protective and/or therapeutic T-cell responses against HBV, including CD4+ and CD8+ T-cell responses. Supporting data are described using mouse models, including HLA-A2 transgenic mice and HBV transgenic mice, with immunogenicity and functional readouts directed to polymerase and core epitopes.
Claims Coverage
The claim set centers on a mutant HBV polymerase domain polypeptide with an internal deletion that functionally disrupts polymerase activity. Additional features refine the sequence constraints, RNaseH-domain disruption options, fusion protein architectures, and pharmaceutical compositions, yielding six inventive features.
Mutant HBV polymerase domain with internal deletion disrupting polymerase activity
A mutant polypeptide comprising a mutated HBV polymerase domain with an internal deletion of at least 4 amino acid residues and at most 30 amino acid residues; the mutated polymerase domain comprises the amino acid sequence shown in SEQ ID NO:1 but lacks at least the Tyr residue at position 203, the Met residue at position 204, the Asp residues at positions 205 and 206, the Val residues at positions 207 and 208, and the Leu residue at position 209.
Mutated polymerase domain defined by sequence identity constraint
The mutant polypeptide in which the mutated polymerase domain comprises an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO:2.
Mutated RNaseH domain with functional disruption options
A mutant polypeptide further including a mutated RNaseH domain with amino-acid deletions or substitutions that functionally disrupt RNaseH activity of a native HBV polymerase, including deletions of at least 8 amino acids and at most 60 amino acids within a defined SEQ ID NO:3 segment and specified substitutions at positions 10, 90, 97, and 98 in any combination.
Fusion with HBV core polypeptide
A fusion protein comprising the mutant polypeptide with the HBV core polypeptide.
Fusion including N-terminal core and one or two HBsAg immunogenic domains
A fusion protein having an N-terminal core polypeptide fused to a mutant polypeptide and including one or two HBsAg immunogenic domains replacing the internal deletions in the mutated polymerase domain and/or in place of the deletion in the mutated RNaseH domain.
Pharmaceutical composition formulated for specified administration routes
The composition formulated for intramuscular, subcutaneous, intradermal administration or scarification.
Overall, the claim coverage centers on internal-deletion mutant HBV polymerase domains that functionally disrupt polymerase activity, with refinements for sequence identity, optional RNaseH-domain disruption, fusion constructs with HBV core polypeptides and HBsAg immunogenic domains, and pharmaceutical compositions for specified administration routes.
Stated Advantages
Eliciting protective and/or therapeutic T-cell responses against HBV.
Documented Applications
Eliciting protective and/or therapeutic T-cell responses against HBV, including CD4+ and CD8+ T-cell responses, supported by immunogenicity and functional data in HLA-A2 transgenic mice and HBV transgenic mice.
Immunogenicity assessment of polymerase and core epitopes in mouse models, including Elispot/ICS results and in vivo CTL lysis.
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