RSV vaccines and methods of production and use thereof
Recombinant, live, attenuated viruses of the Pneumoviridae family are disclosed that include a baculovirus GP64 envelope glycoprotein or variant or fragment thereof and a respiratory syncytial virus (RSV) F protein variant or fragment thereof. Also disclosed are polynucleotides encoding the virus as well as pharmaceutical compositions and vaccines containing the virus. In addition, methods of producing and using each of the above compositions are also disclosed.
1. An isolated polynucleotide encoding at least a portion of an infection-attenuated virus of the Pneumoviridae family, the polynucleotide comprising:
a gene encoding a baculovirus GP64 envelope glycoprotein or variant or fragment thereof; and
a gene encoding an RSV F protein variant or fragment thereof that comprises at least one amino acid substitution compared to a native RSV F protein, wherein the at least one amino acid substitution stabilizes the RSV F protein variant or fragment thereof in a pre-fusion conformation; and
wherein the genome comprises at least one additional modification that renders the virus infection-attenuated.
2. The polynucleotide of claim 1 , wherein the at least one amino acid substitution that stabilizes the RSV F protein variant or fragment thereof in a pre-fusion conformation comprises at least one of the amino acid substitutions S155C, S190F, V207L, and S290C when compared to the native RSV F protein sequence of SEQ ID NO:1.
3. The polynucleotide of claim 1 , wherein the RSV F protein variant or fragment thereof comprises the amino acid substitutions S155C, S190F, V207L, and S290C when compared to the native RSV F protein sequence of SEQ ID NO:1.
4. The polynucleotide of claim 1 , wherein the virus is further defined as a recombinant respiratory syncytial virus.
5. The polynucleotide of claim 1 , wherein the gene encoding the RSV F protein variant or fragment thereof has been codon-optimized.
6. The polynucleotide of claim 1 , wherein the gene encoding the RSV F protein variant or fragment thereof comprises at least one of SEQ ID NOS:5-10.
7. The polynucleotide of claim 1 , further comprising at least one of:
a gene encoding an RSV NS1 protein or a variant or fragment thereof;
a gene encoding an N protein or a variant or fragment thereof;
a gene encoding a P protein or a variant or fragment thereof;
a gene encoding an M protein or a variant or fragment thereof;
a gene encoding an SH protein or a variant or fragment thereof;
a gene encoding a G protein or a variant or fragment thereof;
a gene encoding an M-2 protein or a variant or fragment thereof;
a gene encoding L protein or a variant or fragment thereof; or
any combination thereof.
8. The polynucleotide of claim 1 , wherein:
(a) a portion of the gene encoding the baculovirus GP64 envelope glycoprotein or variant or fragment thereof encodes an ectodomain of the baculovirus GP64 envelope glycoprotein;
(b) a portion of the gene encoding the baculovirus GP64 envelope glycoprotein or variant or fragment thereof encodes an ectodomain and a transmembrane domain of the baculovirus GP64 envelope glycoprotein;
(c) a portion of the gene encoding the baculovirus GP64 envelope glycoprotein or variant or fragment thereof encodes a heterologous cytoplasmic tail; and/or
(d) at least a portion of the gene encoding the baculovirus GP64 envelope glycoprotein or variant or fragment thereof encodes an amino acid sequence represented by SEQ ID NO:15.
9. A mammalian cell, comprising:
at least one polynucleotide encoding a baculovirus GP64 envelope glycoprotein or variant or fragment thereof; and
at least one polynucleotide encoding at least a portion of a genome of an infection-attenuated virus of the Pneumoviridae family, wherein the genome comprises a gene encoding an RSV F protein variant or fragment thereof that comprises at least one amino acid substitution compared to a native RSV F protein, wherein the at least one amino acid substitution stabilizes the RSV F protein variant or fragment thereof in a pre-fusion conformation, and wherein the genome comprises at least one additional modification that renders the virus infection-attenuated; and
wherein the cell produces a recombinant, live, attenuated virus of the Pneumoviridae family.
10. The mammalian cell of claim 9 , further defined as a Vero, HEp-2, or 293 cell.
11. The mammalian cell of claim 9 , further defined as a Vbac cell.
12. The mammalian cell of claim 9 , wherein the at least one polynucleotide encoding a baculovirus GP64 envelope glycoprotein or variant or fragment thereof is separate from and not present in the viral genome.
13. The mammalian cell of claim 9 , wherein the at least one polynucleotide encoding a baculovirus GP64 envelope glycoprotein or variant or fragment thereof is present in the viral genome.
14. The mammalian cell of claim 9 , wherein the at least one amino acid substitution that stabilizes the RSV F protein variant or fragment thereof in a pre-fusion conformation comprises at least one of the amino acid substitutions S155C, S190F, V207L, and S290C when compared to the native RSV F protein sequence of SEQ ID NO:1.
15. The mammalian cell of claim 9 , wherein the RSV F protein variant or fragment thereof comprises the amino acid substitutions S155C, S190F, V207L, and S290C when compared to the native RSV F protein sequence of SEQ ID NO:1.
16. The mammalian cell of claim 9 , wherein the virus is further defined as a recombinant respiratory syncytial virus.
17. The mammalian cell of claim 9 , wherein the gene encoding the RSV F protein variant or fragment thereof has been codon-optimized.
18. The mammalian cell of claim 9 , wherein the gene encoding the RSV F protein variant or fragment thereof comprises at least one of SEQ ID NOS:5-10.
19. The mammalian cell of claim 9 , wherein the at least one polynucleotide encoding at least a portion of the genome of the infection-attenuated virus of the Pneumoviridae family further comprises at least one of:
a gene encoding an RSV NS1 protein or a variant or fragment thereof;
a gene encoding an N protein or a variant or fragment thereof;
a gene encoding a P protein or a variant or fragment thereof;
a gene encoding an M protein or a variant or fragment thereof;
a gene encoding an SH protein or a variant or fragment thereof;
a gene encoding a G protein or a variant or fragment thereof;
a gene encoding an M-2 protein or a variant or fragment thereof;
a gene encoding L protein or a variant or fragment thereof; or
any combination thereof.
20. The mammalian cell of claim 9 , wherein:
(a) a portion of the polynucleotide encoding the baculovirus GP64 envelope glycoprotein or variant or fragment thereof encodes an ectodomain of the baculovirus GP64 envelope glycoprotein;
(b) a portion of the polynucleotide encoding the baculovirus GP64 envelope glycoprotein or variant or fragment thereof encodes an ectodomain and a transmembrane domain of the baculovirus GP64 envelope glycoprotein;
(c) a portion of the polynucleotide encoding the baculovirus GP64 envelope glycoprotein or variant or fragment thereof encodes a heterologous cytoplasmic tail; and/or
(d) at least a portion of the polynucleotide encoding the baculovirus GP64 envelope glycoprotein or variant or fragment thereof encodes an amino acid sequence represented by SEQ ID NO:15.
21. A method of producing recombinant, live, infection-attenuated virus of the Pneumoviridae family, the method comprising the steps of:
culturing the mammalian cell of claim 9 under conditions that allow for production of the recombinant, live, attenuated virus; and
recovering recombinant, live, infection-attenuated virus.
22. The method of claim 21 , further comprising the step of amplifying the attenuated virus in a cell line expressing the baculovirus GP64 envelope glycoprotein or variant or fragment thereof.