Virus-like particles as vaccines for paramyxovirus
The present invention discloses the method of making and using a novel, non-infective, paramyxovirus vaccine. Paramyxovirus structural proteins within a virus-like particle (VLP) comprise one example of such a vaccine. It is observed that the presence of matrix protein, alone, is sufficient and necessary to provide an effective VLP release. Co-expression of four paramyxovirus structural proteins, however, result in the release of non-infective VLPs with densities and efficiencies of release similar to that of infective particles. Representative diseases wherein a VLP vaccine might be useful include, but are not limited to, Newcastle disease, measles, respiratory syncytial virus infection, and parainfluenza 3 virus infection.
1. A non-infectious, replication incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle
i) lacks NDV RNA, and
ii) is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence comprising a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein,
wherein at least 85% of the expressed NDV matrix protein is released from said cell.
2. The virus-like-particle of claim 1 , further comprising a viral glycoprotein.
3. The virus-like particle of claim 2 , comprising at least two viral glycoproteins.
4. The virus-like particle of claim 1 , further comprising one or more NDV proteins selected from the group consisting of nucleocapsid protein, fusion protein, and hemagglutinin-neuraminidase protein.
5. A non-infectious, replication incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle
i) lacks NDV RNA, and
ii) is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence comprising a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein, and
wherein at least 85% of the expressed NDV matrix protein is released from said cell, and wherein said NDV matrix protein comprises a Late Domain that functions in extracellular release of said virus-like particles from said cell.
6. The virus-like-particle of claim 5 , wherein said Late Domain comprises the amino acid sequence PKSP (SEQ ID NO:117).
7. The virus-like-particle of claim 5 , wherein said Late Domain comprises the amino acid sequence YANL (SEQ ID NO:118).
8. A non-infectious, replication incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence comprising a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein, wherein at least 85% of the expressed NDV matrix protein is released from said cell, wherein said NDV matrix protein comprises a Late Domain that functions in extracellular release of said virus-like particles from said cell, and wherein said Late Domain comprises an amino acid sequence selected from the group consisting of YPDL (SEQ ID NO:119) and PTAP (SEQ ID NO:4).
9. The virus-like-particle of claim 1 , wherein said cell is an avian cell.
10. The virus-like-particle of claim 1 , wherein said cell is an insect cell.
11. The virus-like-particle of claim 1 , wherein said virus-like particle is released from said cell at an efficiency of 90%.
12. The virus-like-particle of claim 1 , wherein said virus-like particle is released from said cell at an efficiency of approximately 100%.
13. The virus-like-particle of claim 1 , wherein said Newcastle Disease Virus (NDV) matrix protein is selected from the group consisting of (a) SEQ ID NO:12, (b) SEQ ID NO:24, (c) SEQ ID NO:26, and (d) a protein comprised in an amino acid sequence encoded by nucleotide sequence SEQ ID NO:28.
14. The virus-like-particle of claim 6 , wherein said NDV matrix protein comprises a sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:24.
15. The virus-like particle of claim 7 , wherein said NDV matrix protein comprises a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:24, and SEQ ID NO:26.
16. A non-infectious, replication incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence comprising a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein, wherein least 85% of the expressed NDV matrix protein is released from said cell, wherein said NDV matrix protein comprises a Late Domain that functions in extracellular release of said virus-like particles from said cell, wherein said NDV matrix protein comprises a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:24, and SEQ ID NO:26, except that said amino acid sequence YANL that is at amino acids 232 to 235 of said SEQ ID NO:12, SEQ ID NO:24, and SEQ ID NO:26 is substituted with amino acid sequence PTAP (SEQ ID NO:4).
17. A non-infectious, replication incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence comprising a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein, wherein at least 85% of the expressed NDV matrix protein is released from said cell, wherein said NDV matrix protein comprises a Late Domain that functions in extracellular release of said virus-like particles from said cell, wherein said NDV matrix protein comprises a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:24, and SEQ ID NO:26, except that said amino acid sequence YANL that is at amino acids 232 to 235 of said SEQ ID NO:12, SEQ ID NO:24, and SEQ ID NO:26 is substituted with amino acid sequence YPDL (SEQ ID NO:119).
18. A non-infectious, replication-incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle lacks NDV RNA and is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence consisting of a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein.
19. The virus-like-particle of claim 18 , wherein said virus-like particle is released from said cell at an efficiency greater than the efficiency of release of a VLP comprising NDV M protein, NDV NP protein, NDV F protein, and NDV HN protein.
20. The virus-like-particle of claim 18 , wherein at least 85% of the expressed NDV matrix protein is released from said cell.
21. The virus-like-particle of claim 20 , wherein 90% of the expressed NDV matrix protein is released from said cell.
22. The virus-like-particle of claim 20 , wherein approximately 100% of the expressed NDV matrix protein is released from said cell.
23. The virus-like-particle of claim 19 , further comprising a viral glycoprotein.
24. The virus-like particle of claim 23 , comprising at least two viral glycoproteins.
25. The virus-like particle of claim 19 , further comprising one or more NDV proteins selected from the group consisting of nucleocapsid protein, fusion protein, and hemagglutinin-neuraminidase protein.
26. A non-infectious, replication-incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle lacks NDV RNA and is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence consisting of a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein, wherein said NDV matrix protein comprises a Late Domain that functions in extracellular release of said virus-like particles from said cell.
27. The virus-like-particle of claim 26 , wherein said Late Domain comprises the amino acid sequence PKSP (SEQ ID NO:117).
28. The virus-like-particle of claim 26 , wherein said Late Domain comprises the amino acid sequence YANL (SEQ ID NO:118).
29. A non-infectious, replication-incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence consisting of a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein, wherein said NDV matrix protein comprises a Late Domain that functions in extracellular release of said virus-like particles from said cell, and wherein said Late Domain comprises an amino acid sequence selected from the group consisting of YPDL (SEQ ID NO:119) and PTAP (SEQ ID NO:4).
30. The virus-like-particle of claim 18 , wherein said cell is an avian cell.
31. The virus-like-particle of claim 18 , wherein said cell is an insect cell.
32. The virus-like-particle of claim 18 , wherein said Newcastle Disease Virus (NDV) matrix protein is selected from the group consisting of (a) SEQ ID NO:12, (b) SEQ ID NO:24, (c) SEQ ID NO:26, and (d) a protein comprised in an amino acid sequence encoded by nucleotide sequence SEQ ID NO:28.
33. The virus-like-particle of claim 27 , wherein said NDV matrix protein comprises a sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:24.
34. The virus-like particle of claim 28 , wherein said NDV matrix protein comprises a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:24, and SEQ ID NO:26.
35. A non-infectious, replication-incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence consisting of a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein, wherein said NDV matrix protein comprises a Late Domain that functions in extracellular release of said virus-like particles from said cell, and wherein said Late Domain comprises amino acid sequence PTAP (SEQ ID NO:4).
36. A non-infectious, replication-incompetent virus-like particle comprising a Newcastle Disease Virus (NDV) matrix protein, wherein said virus-like particle is produced by extracellular release from a cell that expresses a recombinant nucleotide sequence consisting of a sequence that encodes said Newcastle Disease Virus (NDV) matrix protein, wherein said NDV matrix protein comprises a Late Domain that functions in extracellular release of said virus-like particles from said cell, and wherein said Late Domain comprises amino acid sequence YPDL (SEQ ID NO:119).
37. A composition comprising the virus-like particle of any one of claims 1 - 10 and 11 - 36 .
38. A method for producing the virus-like particle of claim 1 , 5 , 8 , 16 , 17 , 18 , 26 , 29 , 35 , or 36 , comprising transfecting a cell with one or more expression vector(s) encoding the particle protein(s) under conditions that said cell produces said virus-like particle.
39. A preparation of virus-like particles produced by the method of claim 38 .
40. An isolated cell for producing the virus-like particle of claim 1 , 5 , 8 , 16 , 17 , 18 , 26 , 29 , 35 , or 36 , said cell being transfected with one or more expression vectors encoding the protein(s) necessary to produce said particle.
41. A method for immunizing a host susceptible to Newcastle disease, comprising
a) providing
1) an immunogenic composition comprising a virus-like particle selected from the group consisting of
i) the virus-like particle of claim 1 ,
ii) the virus-like particle of claim 5 ,
iii) the virus-like particle of claim 8 ,
iv) the virus-like particle of claim 16 ,
v) the virus-like particle of claim 17 ,
vi) the virus-like particle of claim 18 ,
vii) the virus-like particle of claim 26 ,
viii) the virus-like particle of claim 29 ,
ix) the virus-like particle of claim 35 , and
x) the virus-like particle of claim 36 , and
2) a host susceptible to Newcastle disease, and
b) administering said immunogenic composition to said host under conditions such that antibodies directed to said virus-like particle are produced.
42. An immunogenic composition comprising a virus-like particle selected from the group consisting of
a) the virus-like particle of claim 1 ,
b) the virus-like particle of claim 5 ,
c) the virus-like particle of claim 8 ,
d) the virus-like particle of claim 16 ,
e) the virus-like particle of claim 17 ,
f) the virus-like particle of claim 18 ,
g) the virus-like particle of claim 26 ,
h) the virus-like particle of claim 29 ,
i) the virus-like particle of claim 35 , and
j) the virus-like particle of claim 36 .
43. A method for producing a virus-like particle selected from the group consisting of
i) the virus-like particle of claim 18 ,
ii) the virus-like particle of claim 26 ,
iii) the virus-like particle of claim 29 ,
iv) the virus-like particle of claim 35 , and
v) the virus-like particle of claim 36 ,
comprising transfecting a cell with one or more expression vector(s) encoding the particle protein(s) under conditions that
a) said cell produces said virus-like particle, and
b) at least 85% of the expressed NDV matrix protein is released from said cell.
44. The method of claim 43 , wherein 90% of the expressed NDV matrix protein is released from said cell.
45. The method of claim 43 , wherein approximately 100% of the expressed NDV matrix protein is released from said cell.