Recombinant parainfluenza virus expression systems and vaccines comprising heterologous antigens derived from metapneumovirus
The present invention relates to recombinant bovine parainfluenza virus (bPIV) cDNA or RNA which may be used to express heterologous gene products in appropriate host cell systems and/or to rescue negative strand RNA recombinant viruses that express, package, and/or present the heterologous gene product. In particular, the heterologous gene products include gene product of another species of PIV or from another negative strand RNA virus, including but not limited to, influenza virus, respiratory syncytial virus, human metapneumovirus and avian pneumovirus . The chimeric viruses and expression products may advantageously be used in vaccine formulations including vaccines against a broad range of pathogens and antigens.
1 . A recombinant parainfluenza virus type 3 comprising a mammalian metapneumovirus nucleotide sequence, wherein the mammalian metapneumovirus is a negative-sense single stranded RNA virus belonging to the sub-family Pneumovirinae of the family Paramyxoviridae and wherein the mammalian metapneumovirus is phylogenetically closer related to a virus isolate deposited as I-2614 with CNCM, Paris than it is related to turkey rhinotracheitis virus (TRTV).
2 . The recombinant parainfluenza virus of claim 1 wherein the mammalian metapneumovirus nucleotide sequence is substituted for a parainfluenza nucleotide sequence or inserted into the parainfluenza virus genome.
3 . The recombinant parainfluenza virus of claim 1 wherein the mammalian metapneumovirus nucleotide sequence is inserted at position 1, 2, 3, 4, 5, or 6 of the parainfluenza virus genome.
4 . The recombinant parainfluenza virus of claim 1 further comprising an RSV nucleotide sequence.
5 . The recombinant parainfluenza virus of claim 1 wherein the parainfluenza virus is a bovine parainfluenza virus.
6 . The recombinant parainfluenza virus of claim 5 further comprising one or more human parainfluenza virus nucleotide sequences.
7 . The recombinant parainfluenza virus of claim 1 wherein the nucleotide sequence encodes a mammalian metapneumovirus polypeptide.
8 . The recombinant parainfluenza virus of claim 7 wherein the polypeptide is the F or G protein of mammalian metapneumovirus , or a fragment thereof.
9 . The recombinant parainfluenza virus of claim 7 wherein the polypeptide is at least 90% identical to SEQ ID NO: 70 or a fragment thereof; is at least 70% identical to SEQ ID NO: 78 or a fragment thereof; is at least 90% identical to SEQ ID NO: 62 or a fragment thereof; is at least 82% identical to SEQ ID NO: 18 or a fragment thereof; is at least 85% identical to SEQ ID NO: 42 or a fragment thereof; is at least 60% identical to SEQ ID NO: 50 or a fragment thereof; is at least 85% identical to SEQ ID NO: 34 or a fragment thereof; is at least 20% identical to SEQ ID NO: 26 or a fragment thereof; or is at least 30% identical to SEQ ID NO: 86 or a fragment thereof.
10 . The recombinant parainfluenza virus of claim 7 wherein the polypeptide is SEQ ID NO: 78, SEQ ID NO: 62, SEQ ID NO: 18, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 34, SEQ ID NO: 26, SEQ ID NO: 86, SEQ ID NO: 70, SEQ ID NO: 28, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 64, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 88, SEQ ID NO: 36, SEQ ID NO: 29, SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO:65, SEQ ID NO:21, SEQ ID NO:45, SEQ ID NO:53, SEQ ID NO:89, SEQ ID NO: 37, SEQ ID NO: 27, SEQ ID NO: 71, SEQ ID NO: 79, SEQ ID NO: 63, SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 87, SEQ ID NO: 35, or a fragment thereof.
11 . The recombinant parainfluenza virus of claim 1 wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO:22-25; SEQ ID NO:30-33; SEQ ID NO:38-41; SEQ ID NO:46-49; SEQ ID NO:54-61; SEQ ID NO:66-69; SEQ ID NO:74-77; SEQ ID NO:82-85; SEQ ID NO:90-93; SEQ ID NO:98-132; SEQ ID NO:168 247; or a fragment thereof.
12 . The recombinant parainfluenza virus of claims 9 or 10 wherein the fragment is at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 250, at least 500, at least 750, or at least 1000 amino acids in length.
13 . The recombinant parainfluenza virus of claim 11 wherein the fragment is at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 250, at least 500, at least 750, or at least 1000 nucleotides in length.
14 . The virus of claim 1 wherein the heterologous nucleotide sequence is derived from a human metapneumovirus.
15 . The virus of claim 7 wherein the mammalian metapneumovirus sequence encodes an F protein, a G protein, an SH protein, an N protein, a P protein, an M2 protein, an M2-1 protein, an M2-2 protein, an L protein, or a fragment thereof.
16 . A recombinant DNA or RNA molecule encoding the genome of the virus of any of claims 1 - 14 .
17 . A recombinant DNA or RNA molecule encoding the genome of the virus of claim 15 .
18 . A vaccine formulation comprising the recombinant virus of any of claims 1 - 14 and a pharmaceutically acceptable excipient.
19 . A vaccine formulation comprising the recombinant virus of claim 15 and a pharmaceutically acceptable excipient.
20 . A method of treating a respiratory tract infection in a mammal, said method comprising administering the vaccine of claim 18 .
21 . A method of treating a respiratory tract infection in a mammal, said method comprising administering the vaccine of claim 19 .
22 . The method of claim 20 wherein the mammal is a human.
23 . The method of claim 21 wherein the mammal is a human.
24 . A method for propagating the recombinant virus of any of claims 1 - 15 , wherein the method comprises culturing cells that are infected with the virus at a temperature wherein the temperature is lower than the temperature that is optimal for growth of the cells.
25 . A method for propagating the recombinant virus of any of claims 1 - 15 , wherein the method comprises (i) culturing cells at a first temperature before infection with the virus; (ii) infecting the cells with the virus; and (iii) culturing the cells at a second temperature after infection of the cells with the virus, wherein the second temperature is lower than the first temperature.
26 . A method for propagating the recombinant virus of any of claims 1 - 15 , wherein the method comprises culturing cells that are infected with the virus in the absence of serum.
27 . A method for propagating the recombinant virus of any of claims 1 - 15 , wherein the method comprises (i) culturing cells in the presence of serum before infection with the virus; (ii) infecting the cells with the virus; and (iii) culturing the cells in the absence of serum after infection of the cells with the virus.
28 . A method for propagating the recombinant virus of any of claims 1 - 15 , wherein the method comprises culturing cells that are infected with the virus without serum at a temperature lower than the temperature that is optimal for growth of the cells.