Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences and methods for propagating virus
AssigneeMedImmune Vaccines, Inc.
InventorJeanne Schickli
Life of this patent
Estimating the term…
Abstract
The invention relates to improved strains of mammalian negative strand RNA virus, metapneumovirus (MPV), within the sub-family Pneumoviridae, of the family Paramyxoviridae. The invention further related to methods for propagating mammalian MPV in the absence of trypsin. The methods and compositions of the invention can be used for the preparation of vaccines against, e.g., MPV infections.
Claims26
A method for propagating mammalian metapneumovirus, wherein the method comprises culturing the mammalian metapneumovirus in medium with a specific trypsin activity of less than 20 milliunits per milliliter of medium.
An isolated mammalian metapneumovirus, wherein the mammalian metapneumovirus is capable of growth in the absence of trypsin.
An isolated nucleic acid, wherein the isolated nucleic acid encodes an F protein of a mammalian metapneumovirus, wherein the F protein comprises the S101P amino acid substitution and at least one of the following amino acid substitutions E93K, Q100K, E92K, E93V, I95S, E96K, Q94K, Q94H, I95S, N97K or N97H.
A method for identifying an F protein of a mammalian metapneumovirus that supports stable growth of the mammalian metapneumovirus in the absence of trypsin, the method comprising:
- (a) growing the mammalian metapneumovirus in the absence of trypsin for at least two passages, wherein the mammalian metapneumovirus comprises a RQPR motif in the cleavage site of the F protein;
- and
- (b) measuring syncytia formation;
- wherein increased syncytia formation relative to syncytia formation by a mammalian metapneumovirus prior to step (a) indicates that the F protein of the mammalian metapneumovirus has acquired an additional amino acid substitution that supports stable growth of the mammalian metapneumovirus in the absence of trypsin.
A method for identifying an F protein of a mammalian metapneumovirus that supports stable growth of the mammalian metapneumovirus in the absence of trypsin, the method comprising:
- (a) growing the mammalian metapneumovirus in the absence of trypsin for at least two passages, wherein the mammalian metapneumovirus comprises a RQPR motif in the cleavage site of the F protein;
- and
- (b) measuring F protein cleavage;
- wherein increased F protein cleavage relative to F protein cleavage by mammalian metapneumovirus prior to step (a) indicates that the F protein of the mammalian metapneumovirus has acquired an additional amino acid substitution that supports stable growth of the mammalian metapneumovirus in the absence of trypsin.
A method for identifying an F protein mutant of a mammalian metapneumovirus that enhances trypsin-independent cleavage of the F protein, wherein the F protein comprises a RQPR motif in the cleavage site, said method comprising:
- (a) growing the mammalian metapneumovirus in the absence of trypsin for at least two passages;
- and
- (b) determining the cleave efficiency of the F protein,
- wherein increased cleavage efficiency of the F protein indicates that the F protein has acquired a mutation that enhances trypsin-independent cleavage of the F protein.
A method for identifying a protease that catalyzes the cleavage of an F protein of mammalian metapneumovirus, wherein the F protein comprises a RQPR motif in the cleavage site, said method comprising:
- (a) contacting the F protein with a test protease;
- and
- (b) determining whether cleavage of the F protein has occurred;
- wherein the occurrence of cleavage of the F protein indicates that the protease catalyzes the cleavage of the F protein.
Dependencies are inferred from the claim text.