Rotavirus particles with chimeric surface proteins
AssigneeNovartis AG
InventorsPhilip R. Dormitzer, Nikolaus Grigorieff, Stephen Harrison, Junhua Pan, Ethan Settembre
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Abstract
The present invention relates to the use of rotavirus particles for displaying a heterologous protein, alone or in complex with another molecule. The invention further relates to methods that employ these modified rotavirus particles to rapidly determine the structure of the heterologous protein or the complex using cryo-electron microscopy (cryo-EM). The invention also relates to a method of immunizing a patient, wherein said method comprises administering to the patient the modified rotavirus particles of the invention.
Claims14
A chimeric protein complex comprising a trimer-forming rotavirus VP7 surface protein linked to a heterologous protein, wherein the rotavirus VP7 surface protein is linked to the heterologous protein non-covalently by a two-part adapter system, wherein the first part of the adapter system comprises a first adapter polypeptide that is fused to the rotavirus VP7 surface protein optionally via a linker sequence, and the second part of the adapter system comprises a second adapter polypeptide that is fused to the heterologous protein optionally via a linker sequence, wherein the first and the second parts of the adapter system form a stable complex with each other, and wherein the chimeric protein complex is capable of recoating and thereby forming a part of an outer layer of double-layered rotavirus particles in vitro.
A nucleic acid composition comprising:
- (a) an open reading frame encoding a modified rotavirus surface protein comprising
- a trimer-forming rotavirus VP7 surface protein,
- a first adapter polypeptide that is fused to the trimer-forming rotavirus VP7 surface protein optionally via a linker sequence;
- and
- (b) an open reading frame encoding a fusion protein comprising
- a protein that is heterologous to the trimer-forming rotavirus VP7 surface protein,
- a second adapter polypeptide that is fused to the heterologous protein optionally via a linker sequence,
- wherein the first and the second adapter polypeptides form a stable protein complex with each other;
- and
- (c) optionally a promoter sequence that is operationally linked to the open reading frame of (a) and further optionally a promoter sequence that is operationally linked to the open reading frame of (b).
A kit comprising:
- (a) a first nucleic acid encoding a modified rotavirus surface protein comprising a trimer-forming rotavirus VP7 surface protein and a first adapter polypeptide, and a second nucleic acid comprising a nucleotide sequence encoding a second adapter polypeptide and a multiple cloning site, and wherein insertion of a coding region for a heterologous protein in the multiple cloning site yields an open reading frame encoding a fusion protein comprising the heterologous protein and the second adapter polypeptide;
- or
- (b) a first nucleic acid encoding a modified rotavirus surface protein comprising a trimer-forming rotavirus VP7 surface protein and a first adapter polypeptide and a second nucleic acid encoding a fusion protein comprising a heterologous protein and a second adapter polypeptide;
- wherein, in each case (a) and (b), the first adapter polypeptide and the second adapter polypeptide are able to form a stable protein complex,
- optionally wherein the kit further comprises a rotavirus particle, wherein the particle is from the same species of rotavirus as the rotavirus from which the trimer-forming VP7 surface protein originated or from a different rotavirus species.
Dependencies are inferred from the claim text.