RSV vaccines and methods of production and use thereof

1. A recombinant, live, attenuated virus of the Pneumoviridae family comprising:

a baculovirus GP64 envelope glycoprotein or variant or fragment thereof, wherein the baculovirus G64 envelope glycoprotein or variant or fragment thereof is capable of mediating entry of the recombinant virus into a mammalian cell; and

a polynucleotide encoding a respiratory syncytial virus (RSV) F protein variant or fragment thereof, wherein the RSV F protein variant or fragment thereof 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.

2. The recombinant, live, attenuated virus of claim 1 , further defined as a recombinant respiratory syncytial virus.

3. The recombinant, live, attenuated virus of claim 1 , wherein the recombinant, live, attenuated virus maintains infective stability when stored at above 0° C. for at least 3.5 days.

4. The recombinant, live, attenuated virus of claim 1 , wherein:

(a) the baculovirus GP64 envelope glycoprotein or variant or fragment thereof comprises an ectodomain of the baculovirus GP64 envelope glycoprotein;

(b) the baculovirus GP64 envelope glycoprotein or variant or fragment thereof comprises an ectodomain and a transmembrane domain of the baculovirus GP64 envelope glycoprotein;

(c) the baculovirus GP64 envelope glycoprotein or variant or fragment thereof comprises a heterologous cytoplasmic tail; and/or

(d) the baculovirus GP64 envelope glycoprotein or variant or fragment thereof comprises an amino acid sequence represented by SEQ ID NO:15.

5. The recombinant, live, attenuated virus of claim 1 , further defined as an enveloped recombinant, live, attenuated virus.

6. The recombinant, live, attenuated virus of claim 1 , wherein the virus is capable of infecting a cell in a mammal but cannot transmit from said cell to another cell in the mammal.

7. The recombinant, live, attenuated virus of claim 1 , wherein:

(a) the RSV F protein variant or fragment thereof is absent at least a portion of a cytoplasmic tail of the native RSV F protein;

(b) the RSV F protein variant or fragment thereof is absent at least a portion of a transmembrane domain and at least a portion of a cytoplasmic tail of the native RSV F protein;

(c) the RSV F protein variant or fragment thereof further comprises at least one of an epitope tag and a detectable marker; and/or

(d) the RSV F protein variant or fragment thereof comprises an amino acid sequence represented by at least one of SEQ ID NOS:2-4.

8. The recombinant, live, attenuated virus of claim 1 , wherein the polynucleotide encoding the RSV F protein variant or fragment thereof has been codon-optimized.

9. The recombinant, live, attenuated virus of claim 1 , wherein the polynucleotide encoding the RSV F protein variant or fragment thereof comprises at least one of SEQ ID NOS:5-10.

10. The recombinant, live, attenuated virus of claim 1 , further comprising 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.

11. The recombinant, live, attenuated virus of claim 1 , wherein the baculovirus GP64 envelope glycoprotein or variant or fragment thereof is not encoded by the viral genome but rather is obtained from a cell line from which the virus is produced.

12. The recombinant, live, attenuated virus of claim 1 , wherein the baculovirus GP64 envelope glycoprotein or variant or fragment thereof is encoded by the viral genome.

13. The recombinant, live, attenuated virus of claim 1 , further encoding at least one of RSV NS1 protein or a variant or fragment thereof; N protein or a variant or fragment thereof; P protein or a variant or fragment thereof; M protein or a variant or fragment thereof; SH protein or a variant or fragment thereof; G protein or a variant or fragment thereof; M-2 protein or a variant or fragment thereof; L protein or a variant or fragment thereof; or any combination thereof.

14. The recombinant, live, attenuated virus of claim 1 , further defined as:

lacking expression of at least one virulence factor encoded by the wild type virus; and/or

lacking expression of secreted G protein (Gmem).

15. An isolated immunogenic composition, comprising:

the recombinant, live, attenuated virus of claim 1 .

16. A pharmaceutical composition, comprising:

a therapeutically effective amount of a recombinant, live, attenuated virus of the Pneumoviridae family comprising:

a baculovirus GP64 envelope glycoprotein or variant or fragment thereof, wherein the baculovirus G64 envelope glycoprotein or variant or fragment thereof is capable of mediating entry of the recombinant virus into a mammalian cell; and

a polynucleotide encoding a respiratory syncytial virus (RSV) F protein variant or fragment thereof, wherein the RSV F protein variant or fragment thereof 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.

17. The pharmaceutical composition of claim 16 , wherein:

(i) the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient;

(ii) the pharmaceutical composition further comprises an adjuvant;

(iii) the pharmaceutical composition is capable of eliciting an immune response against the virus or a component thereof in a mammal; and/or

(iv) the therapeutically effective amount of the recombinant, live, attenuated virus is further defined as an amount sufficient to induce an immune response protective against RSV infection.

18. A method of producing the recombinant, live, attenuated virus of claim 1 , comprising the steps of:

culturing a cell line that expresses a baculovirus GP64 envelope glycoprotein or variant or fragment thereof, the cell line being transfected with at least one polynucleotide encoding RSV virus, wherein the RSV virus comprises 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 cell line is cultured under conditions that allow for production of the recombinant, live, attenuated virus.

19. The method of claim 18 , further defined as comprising the steps of:

recovering recombinant, live, attenuated virus comprising a polynucleotide encoding a respiratory syncytial virus (RSV) F protein variant or fragment thereof from cDNA using reverse genetics in the presence of a baculovirus GP64 envelope glycoprotein or variant or fragment thereof, wherein the RSV F protein variant or fragment thereof 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

amplifying the attenuated virus in a cell line expressing the baculovirus GP64 envelope glycoprotein or variant or fragment thereof.

20. A method of eliciting an immune response in a mammal, comprising the step of:

introducing into the mammal the pharmaceutical composition of claim 16 .