Metapneumovirus strains and their use in vaccine formulations and as vectors for expression of antigenic sequences and methods for propagating virus
View Patent ↗The present invention provides an isolated mammalian negative strand RNA virus, metapneumovirus (MPV), within the sub-family Pneumoviridae , of the family Paramyxoviridae. The invention also provides isolated mammalian negative strand RNA viruses identifiable as phylogenetically corresponding or relating to the genus Metapneumovirus and components thereof. In particular the invention provides a mammalian MPV, subgroups and variants thereof. The invention relates to genomic nucleotide sequences of different isolates of mammalian metapneumoviruses , in particular human metapneumoviruses . The invention relates to the use of the sequence information of different isolates of mammalian metapneumoviruses for diagnostic and therapeutic methods. The present invention relates to nucleotide sequences encoding the genome of a metapneumovirus or a portion thereof, including both mammalian and avian metapneumovirus . The invention further encompasses chimeric or recombinant viruses encoded by said nucleotide sequences. The invention also relates to chimeric and recombinant mammalian MPV that comprise one or more non-native or heterologous sequences. The invention further relates to vaccine formulations comprising mammalian or avian metapneumovirus , including recombinant and chimeric forms of said viruses. The vaccine preparations of the invention encompass multivalent vaccines, including bivalent and trivalent vaccine preparations. The invention also provide methods for propagating virus.
1. An isolated infectious, replicating human metapneumovirus (hMPV) wherein the virus comprises at least one genetic modification resulting in at least one alteration from the RQSR sequence (SEQ ID# 395) at amino acid positions 99 to 102 of the F protein; Glu at amino acid position 749 of the L protein; Met at amino acid position 1094 of the L protein, and Lys at amino acid position 746 of the L protein.
2. The virus of claim 1 , wherein the genetic modification is a deletion, a substitution or an addition.
3. The virus of claim 1 , wherein at least one of the genetic alterations consists of 2 or 3 nucleotide substitutions or deletions per codon.
4. The virus of claim 1 , wherein the amino acid at position 101 of the F protein is a proline.
5. The virus of claim 1 or 4 , wherein the F protein of the virus is cleaved in the absence of trypsin.
6. The virus of claim 1 or 4 , wherein the virus is infectious in the absence of trypsin, as tested by a plaque assay, wherein:
(i) recombinant hMPV is rescued in 293T cells, in OPTIMEM media in the presence of LIPOFECTAMINE; and
(ii) transfected 293T cells and supernatant are collected, frozen, thawed and used to inoculate Vero cells.