Subunit respiratory syncytial virus vaccine preparation

• US 20050089525A1
• Filed: 09/03/2002
• Published: 04/28/2005
• Est. Priority Date: 09/13/2001
• Status: Abandoned Application

• Current Assignee: George Cates, Michel H. Klein, Ray Ooman, Sonia Sanhueza
• Original Assignee: George Cates, Michel H. Klein, Ray Ooman, Sonia Sanhueza
• Inventors: Ooman, Ray, Sanhueza, Sonia, Klein, Michel H., Cates, George
• Application Number:
  US10/488,241
• Publication Number:
  US 20050089525A1
• Time in Patent Office: Days
• US Class Current: 424/186.100
• CPC Class Codes: A61K 2039/55505 Inorganic adjuvants
• CPC Class Codes: A61K 2039/55511 Organic adjuvants
• CPC Class Codes: A61K 2039/55577 Saponins; Quil A; QS21; ISCOMS
• CPC Class Codes: A61K 2039/70 Multivalent vaccine
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/155 Paramyxoviridae, e.g. parainfluenza virus
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 2760/18521 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2760/18522 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2760/18534 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2760/18551 Methods of production or purification of viral material
• CPC Class Codes: C12N 2760/18561 Methods of inactivation or attenuation
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: G01N 33/56983 Viruses

Abstract

The fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV) are isolated and purified from respiratory syncytial virus by mild detergent extraction of the proteins from concentrated virus, loading the protein onto a hydroxyapatite or other ion-exchange matrix column and eluting the protein using mild salt treatment. The F, G and M proteins, formulated as immunogenic compositions, are safe and highly immunogenic and protect relevant animal models against decreased caused by respiratory syncytial virus infection.

Claims

1. A mixture of purified fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV).

2. The mixture of claim 1 wherein said fusion (F) protein comprises multimeric fusion (F) proteins.

3. The mixture of claim 2 wherein, when analyzed under non-reducing conditions, said multimeric fusion (F) protein includes heterodimers of molecular weight approximately 70 kDa and dimeric and trimeric forms.

4. The mixture of claim 1 wherein, when analyzed under non-reducing conditions, said attachment (G) protein comprises G protein of molecular weight approximately 95 kDa and G protein of molecular weight approximately 55 kDa and oligomeric G protein.

5. The mixture of claim 1 wherein, when analyzed by SDS-PAGE under non-reducing conditions, said matrix (M) protein comprises M protein of molecular weight approximately 28 to 34 kDa.

6. The mixture of claim 1 wherein, when analyzed by reduced SDS-PAGE analysis, said fusion (F) protein comprises F₁ of molecular weight approximately 48 kDa and F₂ of molecular weight approximately 23 kDa, said attachment (G) protein comprises a G protein of molecular weight approximately 95 kDa and a G protein of molecular weight approximately 55 kDa, and said matrix (M) protein comprises an M protein of approximately 31 kDa.

7. The mixture of claim 1 wherein said F, G and M proteins are present in the relative proportions of:

F from about 35 to about 70 wt %

G from about 2 to about 30 wt %

M from about 10 to about 50 wt %

8. The mixture of claim 7 wherein, when analyzed by SDS-PAGE under reducing conditions and silver stained, the ratio of F₁ of molecular weight approximately 48 kDa to F₂ of molecular weight approximately 23 kDa is between 1:

1 to about 2;

1 by scanning densitometry.

9. The mixture of claim 7 which is at least about 75% pure.

10. The mixture of claim 1 which is devoid of monoclonal antibodies.

11. The mixture of claim 1 which is devoid of lentil lectin and concanavalin A.

12. The mixture of claim 1 wherein said RSV proteins are non-denatured.

13. The mixture of claim 1 wherein said RSV proteins are from one or both of subtypes RSV A and RSV B.

15. An immunogenic composition comprising an immunoeffective amount of the mixture of claim 1.

16. The immunogenic composition of claim 15 formulated as a vaccine for in vivo administration to a host to confer protection against RSV.

17. The immunogenic composition of claim 15 further comprising at least one adjuvant or at least one immunomodulator.

18. The immunogenic composition of claim 17 wherein the at least one adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, QS21, Quil A or derivatives or components thereof, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid, a muramyl dipeptide, a lipoprotein, polyphosphazene, ISCOM matrix, DC-chol, DDA and bacterial toxins or derivatives thereof.

19. The immunogenic composition of claim 16 wherein the host is a primate.

20. The immunogenic composition of claim 19 wherein the primate is a human.

21. The immunogenic composition of claim 15 further comprising at least one additional immunogen.

22. The immunogenic composition of claim 21 wherein said at least one additional immunogen comprises at least one human parainfluenza virus (PIV) protein selected from the group consisting of PIV-1, PIV-2 and PIV-3.

23. A method of generating an immune response in a host, comprising administering thereto an immunoeffective amount of the immunogenic composition of claim 15.

24. The method of claim 23 wherein said immunogenic composition is formulated as a vaccine for in vivo administration to the host and said administration to the host confers protection against respiratory syncytial virus.

25. A method for producing a vaccine for protection against respiratory syncytial virus (RSV), comprising:

administering the immunogenic composition of claim 15 to a test host to determine the amount of and frequency of administration thereof to confer protection against disease caused by RSV; and

formulating the immunogenic composition in a form suitable for administration to a treated host in accordance with said determined amount and frequency of administration.

26. The method of claim 25 wherein the treated host is a human.

27. A method of producing monoclonal antibodies specific for fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV), comprising:

(a) administering an immunogenic composition of claim 15 to at least one mouse to produce at least one immunized mouse;

(b) removing B-lymphocytes from the at least one immunized mouse;

(c) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;

(d) cloning the hybridomas which produce a selected anti-RSV protein antibody;

(e) culturing the selected anti-RSV protein antibody-producing clones; and

(f) isolating anti-RSV protein antibodies from the selected cultures.

33. A method of determining the presence in a sample of antibodies specifically reactive with a fusion (E) protein, attachment (G) protein or matrix (M) protein of respiratory syncytial virus (RSV), comprising the steps of:

(a) contacting the sample with the mixture of claim 1 to produce complexes comprising a respiratory syncytial virus protein and any said antibodies present in the sample specifically reactive therewith; and

(b) determining production of the complexes.

34. A method of determining the presence in a sample of an F, G or M protein of respiratory syncytial virus, comprising the steps of:

(a) immunizing a subject with the immunogenic composition of claim. 15 to produce antibodies specific for F, G and M proteins of RSV;

(b) contacting the sample with the antibodies to produce complexes comprising any RSV protein present in the sample and said protein specific antibodies; and

(c) determining production of complexes.

35. A diagnostic kit for determining the presence of antibodies in a sample specifically reactive with a fusion (F) protein, attachment (G) protein or a matrix (M) protein of respiratory syncytial virus comprising:

(a) a mixture of claim 1;

(b) means for contacting the immunogenic composition with the sample to produce complexes comprising a respiratory syncytial virus protein and any said antibodies present in the sample; and

(c) means for determining production of the complexes.

14. A coisolated and copurified mixture of non-denatured proteins of respiratory syncytial virus (RSV), consisting essentially of the fusion (F) protein, attachment (G) protein and matrix (M) protein of RSV, wherein the mixture is free from lectins and is free from monoclonal antibodies.

28. A method of producing a coisolated and copurified mixture of proteins of respiratory syncytial virus (RSV), which comprises:

growing RSV on cells in a culture medium;

separating the grown virus from the culture medium;

solubilizing at least the fusion (F) protein, attachment (G) protein and the matrix (M) protein from the separated virus; and

coisolating and copurifying the solubilized RSV proteins.

29. The method of claim 28 wherein said coisolation and copurification are effected by:

loading the solubilized proteins onto an ion-exchange matrix; and

selectively coeluting the F, G and M proteins from the ion-exchange matrix.

30. The method of claim 29 wherein said ion-exchange matrix is a hydroxyapatite matrix.

31. The method of claim 28 wherein said grown virus is washed with urea to remove contaminants without substantial removal of F, G and M proteins prior to solubilization step.

32. The method of claim 29 including contacting said eluted F, G and M proteins with an anion exchange matrix to remove any residual DNA.

36. A mixture of purified fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV) for use as a pharmaceutical substance in a vaccine against disease caused by infection with respiratory syncytial virus.