Delivery and expression of a hybrid surface protein on the surface of gram positive bacteria Number:6,737,521 from the United States Patent and Trademark Office (PTO) owispatent Process is described for the delivery and expression of hybrid surface proteins to the surface of bacteria. The transformed bacteria are useful as vaccines, for the delivery of other active peptides to animal hosts, as diagnostic reagents and for other purposes.<H expression, bacteria, Delivery, and, ex, 6737521.html, Patent, pto, 6737521

Title: Delivery and expression of a hybrid surface protein on the surface of gram positive bacteria

Abstract: Process is described for the delivery and expression of hybrid surface proteins to the surface of bacteria. The transformed bacteria are useful as vaccines, for the delivery of other active peptides to animal hosts, as diagnostic reagents and for other purposes.

Patent Number: 6,737,521 Issued on 05/18/2004 to Fischetti, et al.

Inventors: Fischetti; Vincent A. (West Hempstead, NY), Pozzi; Gianni (Siena, IT), Schneewind; Olaf (New York, NY)
Assignee: The Rockefeller University (New York, NY)

Appl. No.: 08/302,756

Filed: March 7, 1995

PCT Filed: March 12, 1993

PCT No.: PCT/US93/02355

PCT Pub. No.: WO93/18163

PCT Pub. Date: September 16, 1993

Current U.S. Class: 536/23.4 ; 424/184.1; 424/192.1; 424/200.1; 424/244.1; 435/252.3; 435/253.4; 435/320.1; 536/23.7

Current International Class: C07K 14/31 (20060101); C07K 14/195 (20060101); C07K 14/28 (20060101); C07K 14/315 (20060101); C12N 15/74 (20060101); C12N 15/62 (20060101); A61K 39/385 (20060101); C07K 14/025 (20060101); C07K 14/005 (20060101); A61K 39/00 (20060101); A61K 38/00 (20060101)

Field of Search: 424/192.1,200.1,244.1,184.1 435/172.3,252.3,253.4,320.1 536/23.4,23.7 835/27.47,72

References Cited [Referenced By]

U.S. Patent Documents


4722840	February 1988	Valenzuela et al.
4859465	August 1989	Rutter
5124153	June 1992	Beachey et al.
5149532	September 1992	Brunnel
5500353	March 1996	Smit et al.
5616686	April 1997	Fischetti et al.
5707822	January 1998	Fischetti et al.
5733540	March 1998	Lee
5786205	July 1998	Fischetti et al.
5792463	August 1998	Valenzuela et al.
5820860	October 1998	Michel et al.
5821088	October 1998	Darzins et al.
5843444	December 1998	Michel et al.
5847081	December 1998	Michel et al.
5858362	January 1999	Michel et al.
5965390	October 1999	Bjorck et al.
5968521	October 1999	Michel et al.
5968763	October 1999	Fischetti et al.
6063386	May 2000	Dale et al.
6419932	July 2002	Dale

Foreign Patent Documents


0371199	Jun., 1990	EP
0406857	Jan., 1991	EP
0532090	Mar., 1993	EP
8909064	Oct., 1989	WO
9015872	Dec., 1990	WO
9220805	Nov., 1992	WO

Other References

Bessen et al, In: New Perspectives on Streptococcal Infections Zbl. Bakt. Suppl. 22 pp. 200-202, 1992.* .
Hollingshead et al, Infection & Immunity. 55/12: 3237-3239, Dec. 1987.* .
Pozzi et al, J. Bacterial. 170/4: 1969-1972, Apr. 1988.* .
Hruby et al PNAS 88:3190-3194, Apr. 1991.* .
Bessen et al, J. of Immunology 145/4: 1251-1256, Aug. 1990.* .
Pozzi et al, J of Bacteriol. 161/3:909-912, Mar. 1985.* .
Haynes 1993 Science, 260: 1279-1286.* .
Fox 1994, Bio/Technology 12 pp128.* .
Hoffman et al 1987, Science, 237:639-642.* .
Cox 1991, TIBTECH, vol 9:389-394.* .
Hoffman et al 1993 Mol.Immunological Consideration . . . Ed. Good et al pp 149-167.* .
Charoenvit et al 1991, Science, 251:668-671.* .
Dougan et al 1989, J. Gen Microbiol 135:1397-1406.* .
Pan et al, 1995, Nature Medicine 1(5):471-477.* .
Schodel et al 1991, Gene 99:255-259.* .
Schneewind et al, 1993. The EMBO Journal, 12(12):4803-811.* .
Oggioni et al, 1995, Vaccine, 13(8):775-779.* .
Homonylo-McGavin et al, 1996, J. Bacteriology, 178(3):801-07.* .
Pozzi et al, 1994, Vaccine, 12(12):1071-1077.* .
Navarre et al, 1996, J. Bacteriology, 178(2):441-46.* .
Oggioni et al, 1996, Gene, 169:85-90.* .
Fischetti et al 1990, Mol. Microbiol. 4(9):1603-1605.* .
Ikonomidis et al 1995. Vaccines 95. pp 317-326.* .
Greenwood et al, 1991, J. Mol. Biol. 220(4):821-827.* .
Fischetti, 1991, ScientificAmerican; Jun. 1991, 58-65.* .
Uhlen et al. 1983. Gene fusion vectors based on the gene for Streptococcal protein A. GENE 23: 369-378.* .
Poirier et al. 1989. Fibrinogen binding and resistance to phagocytosis of STreptococcus sanguis expressing cloned M protein of S. Pyogenes. Infection and Immunity 57(1):29-35.* .
Fischetti. 1989. Sterptococcal M protein: Molecular design and biological behavior. Clin. Microbiol. Rev. 2(3):285-314.* .
Charbit et al. 1987. Presentation of two epitopes of the preS2 region of hepatitis B virus on live recombinant bacteria. J. Immunology 139:1658-1664.* .
Newton et al. 1991. Expression and immunogenicity of a Sterptococcal M protein epitope inserted in Salmonella flagellin. Infection and Immunity. 59(6):2158-2165.* .
Wu et al. 1989. Expression of immunogenic epitopes of hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella. PNAS USA 86:4726-4730.* .
Hollingshead et al. 1986. Complete nucleotide sequence of Type6 M protein of the group A streptococcus. J. Biological Chem. 261(4):1677-1686.* .
Horinouchi et al. 1982. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide and streptogramin type B antibiotics. J. Bacteriology 150(2):804-814.* .
Schwarz et al. 1985. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314:111-114.* .
Dyson et al. 1989. The human papillomavirus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934-936.* .
Jochmus-Kudielka. 1989. Antibodies against the human papillomavirus type 16 early proteins in human sera: correlation of anti-E7 reactivity with cervical cancer. J. Natl. Cancer Inst. 81(22):1698-1703.* .
Meneguzzi et al. 1991. Immunization against human papillomavirus type 16 tumor cells with recombinant vaccinia viruses expressing E6 and E7. Virology 181:62-69.* .
Fischetti et al. Current Opinion in Biotechnology 4:603-610, 1993.* .
Medaglini et al PNAS, 92:6868-6872, Jul. 1995.* .
Pozzi et al. Infect & Immun. 60(5):1902-1907, May 1992.* .
Oggioni et al. Ed. Balla et al. In: DNA Transfer Gene Expression Microorganism. Proc. Eur. pp. 235-240, 1993.* .
Harrison et al. Res. Microbiol. 141(7-8): 1009-1012, 1990.* .
O'Callaghan et al. Res. Microbiol. 141(7-8):963-969, 1990.* .
Pozzi et al. Res. Microbiol. 143(5):449-57, 1992.* .
Jagusztyn-Krynicka et al, Inf. & Immunity. 61(3): 1004-1015, 1993.* .
Fischetti et al, Science 244(4911):1487-1490, 1989.* .
International Journal of Medical Microbiology, 1992, Gustav Fischer Verlag, "New Perspectives on Streptococci and Streptococcal Infections" p. 350-352. .
Bio/Technology, vol. 9, No. 12, Dec. 1991, Nature America, Inc., P. Fuchs et al., "Targeting Recombinant Antibodies to the Surface of Escherichia coli: Fusion to a Peptidoglycan Associated Lipoprotein", pp. 1369-1372. .
Gene, vol. 104, No. 2, Aug. 15, 1991, F. Breitling et al., "A Surface Expression Vector for Antibody Screening", pp. 147-153. .
Science, vol. 244, Apr. 7, 1989, AAAS, S.M.C. Newton et al., "Immune Response to Cholera Toxin Epitope Inserted in Salmonella flagellin", pp. 70-72. .
Nature, vol. 351, Jun. 6, 1991, C.K. Stover et al., "New Use of BCG for Recombinant Vaccines", pp. 456-460. .
Infection and Immunity, vol. 60, No. 5, May 1992, G. Pozzi et al., "Delivery and Expression of a Heterologous Antigen on the Surface of Streptococci", pp. 1902-1907..

Primary Examiner: Minnifield; Nita
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis, LLP

Parent Case Text

RELATED APPLICATIONS

The present application is a 371 of PCT/US93/02355, filed Mar. 12, 1993; and a continuation-in-part of U.S. application Ser. No. 07/851,082, filed Mar. 13, 1992, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/814,323, filed Dec. 31, 1991, now abandoned, which is a continuation of U.S. application Ser. No. 07/742,199, filed Aug. 5, 1991, now abandoned, which is a continuation of U.S. application Ser. No. 07/522,440, filed May 11, 1990, now abandoned.

Claims

What is claimed is:

1. An isolated and purified DNA molecule encoding a hybrid protein, said hybrid protein comprising a heterologous polypeptide attached to the N-terminal end of the anchor region of a surface antigen of gram positive bacteria, wherein said DNA molecule, when expressed by gram positive bacteria, is expressed as a surface protein, with the anchor region bound to the bacteria and the heterologous peptide exposed on the surface of the bacteria.

2. A plasmid comprising the isolated and purified DNA molecule according to claim 1.

3. A chromosome comprising the isolated and purified DNA molecule according to claim 1.

4. The plasmid pVMB20, deposited at the American Type Culture Collection under the accession number PTA433.

5. The plasmid pVMB21.

6. The S. gordonii strain GP246.

7. An isolated and purified non-pathogenic gram positive bacteria which expresses a hybrid surface protein comprising an anchor sequence which is attached to the bacteria fused to a heterologous polypeptide, wherein said anchor sequence is the anchor sequence of a surface antigen of a gram positive bacteria.

8. The isolated and purified non-pathogenic gram-positive bacteria according to claim 7 wherein the heterologous polypeptide is an enzyme.

9. The isolated and purified non-pathogenic gram-positive bacteria according to claim 7 wherein the heterologous polypeptide is a surface antigen of a mammalian tumor cell.

10. The isolated and purified non-pathogenic gram-positive bacteria according to claim 7 wherein the heterologous polypeptide is a surface antigen of male sperm.

11. The isolated and purified gram-positive bacteria according to claim 7 wherein the heterologous polypeptide is an allergen.

12. The isolated and purified non-pathogenic gram-positive bacteria according to claim 7 wherein the heterologous polypeptide is selected from the group consisting of an antigenic determinant of a surface antigen of a bacteria, an antigenic determinant of a surface antigen of a virus, an antigenic determinant of a surface antigen of a parasite, and an antigenic determinant of a surface antigen of a fungus.

13. The isolated and purified gram-positive bacteria according to claim 7 wherein the anchor sequence is the anchor sequence of a streptococcal M protein.

14. The isolated and purified non-pathogenic gram-positive bacteria according to claim 13 wherein the heterologous polypeptide is selected from the group consisting of an antigenic determinant of a surface antigen of a bacteria, an antigenic determinant of a surface antigen of a virus, an antigenic determinant of a surface antigen of a parasite, and an antigenic determinant of a surface antigen of a fungus.

15. The isolated and purified non-pathogenic gram-positive bacteria of claim 7 wherein the hybrid surface protein is expressed by a plasmid.

16. The isolated and purified non-pathogenic gram-positive bacteria of claim 7 wherein the hybrid surface protein is expressed by a chromosomal gene.

17. Streptococcus gordonii which expresses the hybrid antigen M6:E7.

18. The Streptococcus gordonii according to claim 17, wherein said S. gordonii has been transformed with the plasmid pVMB21.

19. The Streptococcus gordonii according to claim 17, wherein said S. gordonii is S. gordonii strain GP246.

20. A pharmaceutical composition which comprises a pharmaceutically acceptable carrier and a non-pathogenic gram positive bacteria which expresses a hybrid surface protein, said protein being attached to the bacteria at the carboxy terminus of the anchor sequence of a surface antigen of a gram-positive bacteria, the balance of the hybrid surface protein being a heterologous polypeptide, wherein administration of said composition to a mammal results in the production of specific antibodies to said heteroloious polypeptide.

21. The composition according to claim 20 wherein the non-pathogenic gram-positive bacteria expressing a hybrid surface protein is streptococcus.

22. The composition according to claim 20 wherein the nonpathogenic gram-positive bacteria is Streptococcus gordonii.

23. The composition according to claim 22 wherein the nonpathogenic gram-positive bacteria is Streptococcus gordonii transformed to express the hybrid surface protein M6:E7.

24. The composition according to claim 23 wherein the Streptococcus gordonii is transformed by the plasmid pVMB21.

25. The composition according to claim 23 wherein the Streptococcus gordonii is S. gordonii strain GP246.

26. A method of producing antibodies to a polypeptide in animal in need of such treatment which comprises administration of the pharmaceutical composition according to claim 20 to said animal in an amount sufficient to produce antibodies against said polypeptide.

27. A composition of matter which comprises a carrier and a non-pathogenic gram positive bacteria which expresses a hybrid surface protein, said protein being attached to the bacteria at the carboxy terminus of the anchor sequence of a surface antigen of a gram-positive bacteria, the balance of the hybrid surface protein being a heterologous polypeptide which comprises the antigenic determinant of a surface antigen of a pathogenic bacteria.

28. The composition according to claim 27 wherein the antigenic determinant consists of the antigenic determinant of a surface antigen of a streptococcus.

29. The composition according to claim 27 wherein the nonpathogenic gram-positive bacteria is Streptococcus gordonii.

30. The composition according to claim 27 wherein the nonpathogenic gram-positive bacteria is Streptococcus gordonii transformed to express the hybrid surface protein M6:E7.

31. The composition according to claim 30 wherein the Streptococcus gordonii is transformed by the plasmid pVMB21.

32. The composition according to claim 30 wherein the Streptococcus gordonii is S. gordonii strain GP246.

33. A composition of matter comprising a non-pathogenic gram positive bacteria and a carrier therefor, wherein said bacteria expresses a hybrid surface protein, said hybrid surface protein being attached to the bacteria at the carboxy terminus of the anchor sequence of a surface antigen of a gram-positive bacteria, the balance of the hybrid surface protein being a heterologous polypeptide.

34. An isolated and purified DNA molecule encoding a hybrid protein, said hybrid protein comprising a heterologous polypeptide attached to the N-terminal end of the anchor region of a surface antigen of gram positive bacteria, wherein said anchor region comprises the amino acid sequence Leu-Pro-X-Thr-Gly-X, a hydrophobic domain, and a charged tail.

35. The isolated and purified DNA molecule according to claim 34, wherein said hydrophobic domain is from 15-20 amino acids long.

36. The isolated and purified DNA molecule according to claim 34, wherein said anchor region further comprises a spacer segment between the Leu-Pro-X-Thr-Gly-X sequence and the hydrophobic domain.

37. The isolated and purified DNA molecule according to claim 36, wherein said spacer segment is from 3 to 6 amino acids long.

38. The isolated and purified DNA molecule according to claim 34 wherein said charged tail is from 4 to 6 amino acids long.

39. The isolated and purified DNA molecule according to claim 34, wherein said anchor region consists of the amino acid sequence Leu-Pro-X-Thr-Gly-X, a spacer segment of 3-6 amino acids, a hydrophobic domain of 15-20 amino acids, and a charged tail of 4 to 6 amino acids.

40. The isolated and purified DNA molecule according to claim 34, wherein said anchor region consists of the amino acid sequence Leu-Pro-X-Thr-Gly-X, a spacer segment of 3 amino acids, a hydrophobic domain of 20 amino acids, and a charged tail of 6 amino acids.

41. The isolated and purified DNA molecule according to claim 40, wherein said anchor region consists of the amino acid sequence Leu-Pro-Ser-Thr-Gly-Glu-Thr-Ala-Asn-Pro-Phe-Phe-Thr-Ala-Ala-Ala-Leu-Thr-Val -Met-Ala-Thr-Ala-Gly-Val-Ala-Ala-Val-Val-Lys-Arg-Lys-Glu-Glu-Asn (SEQ ID NO: 1).

42. A plasmid comprising the isolated and purified DNA molecule according to claim 34.

43. A composition of matter comprising the isolated and purified DNA molecule according to claim 34 and a carrier therefor.

44. An isolated and purified microorganism transformed with the isolated and purified DNA molecule according to claim 34.

45. The isolated and purified microorganism according to claim 44 wherein said isolated and purified microorganism is a bacteria.

46. The isolated and purified microorganism according to claim 45 wherein said bacteria is gram-positive.

47. A composition of matter comprising the isolated and purified microorganism according to claim 44 and a carrier therefor.

Description

FIELD OF THE INVENTION

This invention relates generally to products and processes useful to deliver proteins, including protein antigens, to the surface of gram-positive bacteria and firmly attach them to the cell. More specifically, it relates to the production of a fusion protein containing at least the anchor region of the cell associated region of a gram-positive surface protein and any protein, peptide or polypeptide that may be usefully administered to an animal host. The products of the invention which comprise proteins, peptides or polypeptides, as further discussed below, placed on the surface of a gram positive bacteria may be used to deliver such materials to the animal host to elicit an immungenic response, for example the production of protective antibodies or for another useful purpose. The protein, peptide or polypeptide may, for example, be an enzyme or other functional protein necessary for a specific purpose. It may be an antigenic determinant from a bacteria, virus, parasite or fungus. It may be a surface antigen from a mammalian tumor cell or of male sperm. It may be an allergen such as a vespid venom. The invention also relates to novel plasmids, genes, chromosomes and transformed bacteria employed in the production of such fusion proteins. The invention, additionally, provides novel vaccines which employ gram-positive bacteria designed to deliver to an animal host a foreign antigen normally present on a pathogenic microorganism which is associated with the virulence of that pathogen and which will elicit antigens to protect the host against infection or disease caused by the pathogenic microorganism.

The products of the invention are also useful as diagnostic agents.

The invention also provides a means to deliver enzymes, placed on the bacterial surface, to specific areas of interest.

The term "animal" as used herein refers to living beings including mammals such as man; bovines, especially beef cattle; sheep and goats; poultry, especially chickens, ducks and turkeys; as well as fish, especially those raised in fish farms such as salmon, trout and catfish. This invention is of special importance to mammals.

BACKGROUND OF THE INVENTION

The essence of this invention is that it provides a method for the production of novel non-pathogenic gram positive bacteria expressing a hybrid surface protein which may be a hybrid surface antigen, comprising two principal parts, an anchor segment comprised of amino acid residues and an N-terminal active polypeptide segment, both of which will be defined and discussed in more detail below.

This invention will be better understood by consideration of the M protein and its structure. The M protein is a coiled coil surface antigen which is the virulence factor of group A streptococci, a gram positive bacteria. The M protein of Streptococcus pyogenes of M type 6 contains 441 amino acid residues.

Its structure will be discussed in more detail below, but it will be useful to discuss the cell associated region at this point. Using the standard one letter representation of amino acids, the structure of the anchor region of the cell associated region of the M6 protein from amino acid residue 407 to residue 441 may be represented as: LPSTGETANPFFTAAALTVMATAGVAAVVKRKEEN (SEQ ID NO:1)

Reading from the first leucine residue (L) at the N-terminal of the anchor region, the region includes the LPSTGE segment (SEQ ID NO:36); a spacer segment containing three amino acid residues TAN (SEQ ID NO:60); a hydrophobic segment of twenty amino acids, PFTAAALTVMATAGVAVV (SEQ ID NO:2); followed by a highly charged tail segment, KRKEEN (SEQ ID NO:61).

It has been observed as a result of structural studies of a large number of surface proteins of gram positive bacteria that the above described anchor region of the M6 protein is highly conserved among all known surface proteins of gram positive bacteria. Many of them are shown by Fischetti et al (Reference 1). Generally, the hydrophobic segment contains about 15 to 20 amino acid residues, the charged tail segment about 4 to 6 amino acid residues, and the spacer segment from about 3 to 6 amino acid residues. Most remarkable however, is the high degree of homology, practically 100% in the LPSTGE (SEQ ID NO:36) segment of the known surface proteins. The variations that occur are almost exclusively at the 3 and 6 positions. Therefore, the region may be generally represented as LPXTGX (SEQ ID NO:37).

The following Table 1 shows the remarkable extent of this homology thus far established amongst forty different surface proteins of gram positive bacteria.

TABLE 1 [SEQ ID NOS: 36, 38-55, AND 59] SEQUENCED SURFACE PROTEINS FROM GRAM-POSITIVE BACTERIA SEQ NAME/ SURFACE ID GENE PROTEIN ORGANISM LPSTGE REF NO. 1. M6 M protein S. pyogenes LPSTGE (2) 36 2. M5 M protein S. pyogenes LPSTGE (3) 36 3. M12 M protein S. pyogenes LPSTGE (4) 36 4. M24 M protein S. pyogenes LPSTGE (5) 36 5. M49 M protein S. pyogenes LPSTGE (6) 36 6. M57 M protein S. pyogenes LPSTGE (7) 36 7. M2 M protein S. pyogenes LPSTGE (8) 36 8. ARP2 IgA binding S. pyogenes LPSTGE (8) 36 protein 9. ARP4 IgA binding S. pyogenes LPSTGE (9) 36 protein 10. FcRA Fc binding S. pyogenes LPSTGE (10) 36 protein 11. Prot H Human IgG S. pyogenes LPSTGE (11) 36 Fc binding 12. SCP C5a peptidase S. pyogenes LPTTND (12) 38 13. T6 Protease S. pyogenes LPSTGS (13) 39 resistant protein 14. bac IgA binding Gr. B strep LPYTGV (14) 40 protein 15. Prot G IgG binding Gr. G strep LPTTGE (15) 41 protein 16. PAc Surface protein S. mutans LPNTGE (16) 42 17. spaP Surface protein S. mutans LPNTGE (17) 42 18. spaA Surface protein S. sobrinus LPATGD (18) 43 19. wapA Wall-associated S. mutans LPSTGE (19) 36 protein A 20. Sec10 Surface protein E. fecalis LPQTGE (20) 44 21. Asc10 Surface protein E. fecalis LPKTGE (20) 59 22. asa1 Aggregation E. fecalis LPQTGE (21) 44 substance 23. Prot A IgG binding S. aureus LPETGV (22) 45 protein 24. FnBP Fibronectin S. aureus LPETGG (23) 46 binding protein 25. wg2 Cell wall S. cremoris LPKTGE (24) 59 protease 26. In1A Internalization L. monocyto- LPTTGE (25) 47 protein genes 27. Fim- Type 1 A. viscosis LPLTGA (26) 48 briae fimbriae 28. Fim- Type 2 A. naeslundii LPLTGA (27) 48 briae fimbriae 29. Mrp4 IgG/ S. pyogenes LPSTGE (10) 36 Fibrinogen binding 30. sof22 Serum opacity S. pyogenes LPASGD (54) 49 factor 31. Sfb Fibronectin S. pyogenes LPATGD (55) 43 binding 32. Prot L Light chain P. magnus LPKAGS (56) 50 binding 33. bca alpha antigen Gr. B strep LPATGE (57) 51 34. fnbA Fibronectin S. dysgalactiae LPQTGT (58) 52 binding 35. fnbB Fibronectin S. dysgalactiae LPAAGE (59) 53 binding 36. EmmG1 Mprotein Gr. G Strep LPSTGE (60) 36 37. DG12 Albumin Gr. G strep LPSTGE (61) 36 binding protein 38. MRP Surface protein S. suis LPNTGE (62) 44 39. FnBp Fibronectin S.aureus LPETGG (63) 54 binding protein 40. cna Collagen S. aureus LPKTGM (64) 55 binding protein

It is apparent that this highly homologous region of the surface proteins of gram-positive bacteria is essential to anchoring bacterial surface proteins to the cell (28). This segment, which is referred to herein as the LPXTGX segment (SEQ ID NO: 37), is the crucial segment of the cell associated region of surface protein for anchoring the proteins to the surface of gram positive bacteria.

This discovery has been confirmed by Schneewind et al (65). Using the Protein A molecule of Staphylococcus aureus, these investigators have established that the complete complex (LPXTGX motif (SEQ ID NO:37), hydrophobic domain and charged tail) are necessary to deliver the Protein A molecule to the cell surface and that changes in the LPXTGX motif (SEQ ID NO:37) or deletion thereof will not inhibit expression of the molecule but will prevent it from anchoring to the surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the alignment of the amino acids in the C-terminal regions of a variety of surface antigens from gram-positive bacteria and the gene segments which are employed to express them. The LPSTGE motif (SEQ ID NO:36) is shaded and the proteins were aligned along this consensus sequence. In 10 out of 11 proteins, a conserved lysine residue was found 2 or 3 residues preceding the consensus LPSTGE sequence (SEQ ID NO:36) (boxed). The homologous carboxyterminal hydrophobic regions are also boxed. Abbreviations used in the left column are the same as those in Table 1.

FIG. 2 a model of the M protein.

FIG. 3 shows the complete amino acid sequence of the M6 protein.

FIG. 4 shows the host vector system GP232-pVMB20.

FIG. 5 shows the construction and chromosomal integration of the M6:E7 translation fusion in GP246.

FIG. 6 shows the results of studies designed to demonstrate that the M6:E7 fusion protein expressed in Streptococcus gordonii GP246 located on its surface.

FIG. 7 shows that cell extracts of recombinant Streptococcus gordonii246 expresses the M6:E7 fusion protein.

FIG. 8 shows that animals immunized with the recombinant GP246 produce antibodies reactive with the E7 protein.

FIG. 9 shows a universal plasmid of the invention.

FIG. 10 shows a typical hybrid protein of the invention.

FIGS. 11 and 12 show the results of studies in which mice were treated with a hybrid protein of the invention.

Careful study of FIGS. 2 and 3 will assist in understanding this invention. FIG. 2 represents a model of the M protein with certain of the positions and segments identified. FIG. 3 identifies each amino acid residue in the complete structure of the M6 protein.

It will be understood from the foregoing discussion that the segment of the M protein identified as the cell associated region in FIG. 2 is analogous to regions for surface proteins found on other gram-positive bacteria such as those listed in Table 1. As discussed above, there is a high degree of homology particularly within the anchor sequence especially the LPXTGX segment (SEQ ID NO:37) found in all gram-positive surface proteins. As will be described below, and for the purpose of illustrating this invention, the gene segment spanning region 122 to 300 of the gene which expresses the M protein is genetically removed and replaced by a new gene segment expressing a foreign protein, for example, an antigen which will generate useful antibodies, thereby producing a novel hybrid surface protein. Because of the great degree of homology within the cell associated regions of all gram-positive bacteria, the hybrid gene can be employed in any gram-positive bacteria. The selected bacteria will express the desired hybrid protein using the anchor region to attach the molecule to the cell and positioning the inserted active segment on the cell surface. The inserted active segment is hereinafter referred to as the "active polypeptide".

The term "active polypeptide" is used herein in the broadest possible sense. It refers to any peptide, polypeptide or protein which may be delivered to an animal host for any useful purpose. For example, as described in detail hereinafter, the cell associated region of a protein from gram-positive bacteria can be fused to a segment of a viral protein from a pathogen to produce a hybrid surface protein which will be expressed by non-pathogenic bacteria. The bacteria can colonize an animal host and function as a vaccine. It will elicit antibodies in an animal host to protect against or inhibit subsequent infection by the virus. The fused viral segment is the "active polypeptide" of that particular embodiment of the invention.

For convenience, the term "polypeptide" will hereinafter be used to refer to molecules which are of sufficiently high molecular weight to be called proteins, to products of lesser molecular weight, usually called polypeptides, and to products of even lesser molecular weights normally referred to as peptides.

The "active polypeptide" may be any polypeptide which can be delivered to an animal host for a useful purpose. It could be, for example the viral segment referred to above. It could also be an antigen, from any pathogenic virus, or from a bacteria, parasite or fungi. In such instances, the active polypeptide may be the complete antigen, the antigenic determinant of the antigen or a segment of the antigen which includes the antigenic determinant. The useful purpose will be to elicit a protective immune response by the production of antibodies or to elicit a cellular immune response to the antigen.

The term "cell associated region" as used in this specification and claims will be readily understood by reference to the figures, especially FIGS. 2, 3 and 10. It will be seen that the cell associated region includes a cell wall spanning sequence of amino acid residues and a carbohydrate spanning segment. The wall spanning sequence may be omitted from the hybrid proteins of this invention, although it is presently preferred not to do so.

The cell associated region also includes the anchor region which contains the anchor segment (LPXTGX (SEQ ID NO:37)), the spacer segment, the hydrophobic segment and the charged tail segment. The anchor sequence is essential to the hybrid proteins of this invention. Without this sequence, the hybrid protein will not be retained on the surface of the gram positive bacterial carrier.

FIG. 10 shows the balance of a typical hybrid protein of the invention which, as shown, includes the active polypeptide fused to the cell associated region. The figure also shows a leader segment and a small N-terminal segment at the amino terminal of the active polypeptide. These segments cooperate in the proper placement of the hybrid protein on the surface of the gram positive bacteria. The function of the leader segment is to permit the proper processing of the hybrid protein within the cell. The N-terminal segment in cooperation with the leader segment enables the leader peptidase enzyme to separate the leader segment from the N-terminal segment and permit the hybrid protein to assume its proper position on the surface of the bacteria. The leader segment and the N-terminal segment are from the same surface protein as the protein of the cell associated region. The N-terminal segment suitably contains the first few amino acid residues derived from the amino end of the protein used in the cell associated region of the hybrid protein. About ten such residues are normally sufficient for the proper processing, but segments containing up to twenty or more amino acid residues may be employed.

The active polypeptide is not necessarily an antigen from a pathogenic organism. It may also be an enzyme, for example. In that event, the useful purpose will be to deliver the enzyme on the surface of a non-pathogenic bacteria to a specific site in the animal host colonized by that non-pathogenic bacteria.

The active polypeptide may be the complete molecule which comprises the enzyme. Alternatively, it may be only that segment of the polypeptide which is required to accomplish the useful purpose.

The active polypeptide may be an antigen which will react with antibodies elicited by pathogenic microorganisms. Bacteria of the invention carrying such active polypeptide antigens are useful as diagnostic reagents.

When reference is made herein to non-pathogenic bacteria it should be understood to include gram positive commensal bacteria as well as pathogenic bacteria which have been modified or attenuated by the usual procedures so that their pathogenicity is weakened or destroyed and which express hybrid proteins in accordance with the process described herein, such hybrid proteins containing a C-terminal region including the LPXTGX region (SEQ ID NO:37).

BRIEF DESCRIPTION OF THE INVENTION

This invention provides non-pathogenic gram positive bacteria which express at least one hybrid surface protein the C-terminal region of which is attached to the bacteria. The C-terminal region includes, but is not limited to, the cell associated region of a surface protein normally expressed by a gram-positive bacteria which may be pathogenic or non-pathogenic. The balance of the hybrid surface protein includes an active polypeptide which may be delivered to an animal host for any useful purpose. The surface protein "normally produced" by the bacteria of this invention refers to the whole protein produced by the bacteria before it is genetically altered in accordance with the process of this invention.

As is known from previous studies (29) and indicated in FIGS. 2 and 3, amino acids 298-441 of the M protein are buried within the cell whereas residues 1-297 are exposed on the surface of the bacterial cell. In accordance with this invention, by employing genetic manipulations described and illustrated herein, nearly all of the surface exposed region of the M protein may be removed and replaced by an active polypeptide that is linked to the C-terminal cell-associated region of the M protein to produce a hybrid surface protein of the invention. Because the cell associated region is essentially the same for all gram-positive surface molecules (FIG. 10), a smimilar strategy may be used whereby the cell associated region of any one of these molecules may be used as a delivery carrier or vehicle to deliver an active polypeptide from another source. The new gene employed to express the hybrid surface protein may be inserted into a plasmid by standard techniques and the plasmid used to transform an Escherichia coli or other vector. The E. coli thereafter will express the new fusion protein, i.e. the hybrid protein of this invention. The E. coli procedure is useful for the production of large amounts of hybrid protein which can be isolated from the periplasm of this gram negative organism. However, for most utilities of this invention, it is preferred to transform a gram positive organism for the production of the hybrid protein on the surface of the gram positive bacteria. The recombinant genes produced in accordance with the invention may be processed by any gram positive bacteria.

Alternatively, the newly constructed plasmid containing the fusion gene may be used to integrate the fusion gene into the chromosome of a gram-positive bacteria, for example the chromosome of Streptococcus mutans. The newly produced strain of Streptococcus mutans will thereafter produce the new hybrid protein of this invention by expressing it on the cell surface. This is the presently preferred procedure for producing the products of this invention.

Antigenic polypeptides from a wide variety of microorganisms may be employed as the active polypeptides of the invention. These include pathogenic microorganisms which infect man and animals. There follows a representative list of typical microorganisms which express polypeptides useful in the practice of this inventions. The transformed bacteria of the invention may be used to treat or prevent the diseases associated with infection by the microorganism.

Fungi: Candida albicans, Aspergillus fumigatus, Histoplasma capsulatum (all cause disseminating disease), microsporum canis (animal ringworm) Parasitic Protozoa: Plasmodium falciparum (malaria), Trypanosoma cruzi (Sleeping sickness),

Spirochetes: Borrelia bergdorferi (Lyme disease), Treponema pallidum (syphilis), Borrelia recurrentis (recurring fever), Leptospira icterohaemorrhagiae (leptospirosis)

Bacteria: Neisseria gonorrhoeae (gonorrhoea), Staphylococcus aureus (endocarditis), Streptococcus pyogenes (rheumatic fever), Salmonella typhosa (salmonellosis), Hemophilus influenzae (influenza), Bordetella pertussis (whooping cough), Actinomyces israelii (actimomyosis), Streptococcus mutans (dental caries)

Streptococcus equi--Strangles (Horses), Streptococcus agalactiae (bovine mastitis), Streptococcus anginosus (canine genital infections)

Viruses: Human immunodeficiency virus (HIV), Polio virus, Influenza virus, Rabies virus, Herpes virus, Foot and Mouth Disease virus, Psittacosis virus, Paramyxovirus, Myxovirus, Coronovirus

In one embodiment of this invention, a non-pathogenic gram-positive bacteria that is a commensal organism for the host animal will be used to express the hybrid protein on its surface, by inserting the gene coding for the hybrid protein into the non-pathogenic gram-positive bacteria. If the fusion is with an active polypeptide that is an antigen from a pathogenic organism (bacterial, viral, parasitic, or fungal), and the resulting commensal bacteria expressing the hybrid antigen is administered to an animal host, there will be an immunological response by both humoral and cell mediated pathways. One possible immunological response is the production of antibodies, thereby effecting protection against infection by the pathogen. If the fusion is with an enzyme, the enzyme will be expressed on the surface of the commensal. A wide variety of active polypeptides may be delivered to the surface of gram positive. bacteria as will be apparent to the skilled artisan from a study of this disclosure.

The genes, plasmids, chromosomes, and transformed bacteria of this invention are produced by the application of standard recombinant techniques well known to those skilled in the art. For example, an oligonucleotide or a restriction fragment coding for a viral coat protein, the virulence factor of a bacterial surface antigen (or, in fact, the complete antigen), an enzyme or other desired progenitor of a hybrid surface protein of the invention may be ligated to the gene which expresses the cell associated region of the M protein or other known surface protein from a gram positive bacteria such as those listed in Table 1. The resulting hybrid gene will be used to transform a gram positive bacteria to effect the production of a surface hybrid protein of the invention.

This invention will be further explained in conection with the expression of the hybrid protein M6:E7 by the GP246 strain of Streptococcus gordonii. This hybrid antigen contains the C-terminal cell associated segment of the M6 protein. The antibody eliciting polypeptide, E7, is an early protein from a papillomavirus strain.

Streptococcus gordonii is a commensal bacteria of the oral cavity. E7 is a 98 amino acid early protein produced during viral replication of human papillomavirus type 16 (HPV16). It is an oncoprotein, antibodies to which are found in patients with cervical cancer. It is considered a major candidate antigen for vaccines against HPV-induced malignant neoplasias.

The novel bacteria of this invention may be produced by standard homologous recombination shown in FIGS. 4 and 5. As illustrated, a new strain of Streptococcus gordonii, GP232 which expresses large quantitites of M6 protein was employed as the host to produce a new strain GP246.

The procedure employed as illustrated in FIGS. 4 and 5 is standard and widely utilized by the skilled practitioner. The technology, as illustrated, is to construct a gram positive recipient organism for the insertion of the fusion gene in a random location in the chromosome that results in a high expression of the fusion gene. The insertion site may vary from organism to organism and even within the same organism. The advantage of the method is that it insures that the inserted gene is in a site which will result in high expression of the fusion gene, rather than rely on the expression as a result of a foreign promoter. Thus, the method assures that a region with a strong promoter is selected.

In the process of producing the novel GP246 strain, the 538 base pair segment between sites KpnI and HindIII of the M6 protein was excised from the novel plasmid pVMB20, produced as described below, and the 294 base pair encoding for E7 was inserted in its place to produce the novel gene which was utilized for the production of the new plasmid pVMB21. The region of the M protein that was excised results in the placement of the E7 molecule at the cell surface with a 120 amino acid segment of the M6 N-terminus including the leader segment fused to the N-terminus of E7. The construct was made in E. coli and the recombinant plasmid was used to transform S. gordonii by the standard double cross-over procedure with GP232 to effect chromosomal integration of the gene which expresses the M6:E7 protein by the new strain GP246.

MATERIALS AND METHODS

Recombinant DNA techniques. Gene fusions in Escherichia coli vectors were obtained and constructed by standard procedures (30).

Streptococcal transformation. Frozen cells of naturally competent S. gordonii Challis were prepared and transformed by known procedures (31). Plating and scoring of transformants on multilayered plates was performed using erythromycin at a concentration 5 ug/ml in the overlay using known procedures (32).

Genetic analysis of transformants. Transformants were streaked on the surface of 3 blood agar plates by toothpick transfer of colonies from the selection plate. The first plate contained erythromycin (5 ug/ml), the second chloramphenicol (5 ug/ml), and the third had no antibiotic. Plates were incubated 36 hours at 37.degree. C. After incubation, a nitrocellulose membrane was applied to the surface of the plate that contained no antibiotic, and kept for 20 min. at room temperature. The membrane was then incubated 30 min at 37.degree. C. and 15 min at 80.degree. C. in a vacuum oven. The presence of E7 protein bound to the nitrocellulose was detected using anti-E7 polyclonal antibodies (1:5000 dilution), obtained from rabbits immunized with a MS2:E7 fusion protein produced in E. coli (33).

Immunofluoreseence. Bacteria grown in Todd-Hewitt broth (Difco) were harvested in late exponential phase, applied on a glass slide, and fixed with methanol. Slides were treated with M6- or E7-specific antibodies (1:50 dilution), extensively washed, and then reacted with goat anti-rabbit (or anti-mouse) IgG antiserum (1:100 dilution), conjugated with rhodamine. Results were observed under a Con-focal Fluorescence Imaging System MRC-500 Bio-Rad microscope.

Western blot analysis of cell extracts. Streptococci were grown to late stationary phase in Todd-Hewitt borth (Difco). Cells were harvested and resuspended in 50 mM Tris(pH 8.0), 50 mM MgCl.sub.2, 30% sucrose. Protoplasts were obtained by treating the cell suspension with lysozyme (100 ug/ml) for 30 min. at 0.degree. C. Protoplasts were then centrifuged and resuspended in 50 mM Tris (pH 8.0). Thorough lysis was achieved by five cycles of quick freezing/thawing of the suspension. Cells that did not lyse and gross debris were discarded by low speed centrifugation at 1,000 rpm for 15 min, whereas the supernatant, containing membranes and cytoplasm, was used for Western blot analysis. The extract obtained from about 5.times.10.sup.8 streptococcal cells was run on a gel, and Western blot was performed by convenitonal methods.

Immunization of nice. Balb/c mice were immunized subcutaneously with 5.times.10.sup.8 live GP246 streptococcal cells (5.times.10.sup.7 colony forming units) emulsified in complete Freund's adjuvant. Two and three weeks after the primary immunization, animals were given subcutaneous boosters of the same bacterial dose emulsified in incomplete Freund's adjuvant. Animals were bled a week after the last boost.

RESULTS

Production of the plasmid pVMB3: A construct was made where ermC, a gene conferring resistance to erythromycin (34), was cloned adjacent to emm-6.1, so that both genes would be within the same ClaI fragment. In this construct the initiation codon of emm-6.1was 19 bp downstream of one of the ClaI sites, so that ClaI cleavage would leave emm-6.1 promoterless. Plasmid pVV3:M6 (35), containing this ClaI site upstream of the emm-6.1coding sequence, was used in the experiments. The 2.0 kb MspI fragment of pE194, containing ermC, was ligated with a partial ClaI digestion of pVV3:M6. After transformation of E. coli DH5, a clone was isolated with a plasmid, pVMB3, contaiing the ClaI fragment.

Production of the strain GP230: The 3.4-kb ClaI fragment of pVMB3, containing ermC and the promoterless emm6.1, was ligated with chromosomal DNA of S. gordonii also cut with ClaI. The ligation mixture was used to transform the naturally transformable S. gordonii "Challis", strain V288. By this method the emm-6.1/ermC ClaI fragment was integrated at random into the chromosome. The chromosomal DNA ligated to the ClaI fragment provided the homology for integration during transformation. Erythromycin-resistant (Em-r) transformants were selected and analyzed for production of M6 protein by "streak blots". Of 700 Em-r transformants, 196 (28%) produced M6 protein. Based on the semiquantitative dot blot analysis, GP230 appeared to be the best M6 producer.

Production of the strain GP232: S. gordonii GP230 was transformed with the chromosomal DNA of pneumonococcal strain GP69. This pneumonoccal strain is resistant to chloramphenicol but susceptible to erythromycin produced according to the procedure of Pozzi and Guild (36). When GP69 chromosomal DNA is used to transform GP230, recombination occurs at the level of the ermC sequence leading to insertion of the CAT sequence into the copy of ermC integrated into the GP230 chromosome. This insertion yields GP232 which, as discussed and shown in the figures, expresses M6 on its surface and is also resistant to chloramphenicol and sensitive to erythromycin. S. gordonii strain GP232 has been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, pursuant to the terms of the Budapest Treaty, and assigned Accession No. PTA434.

Expression of E7 protein of KPV16 in S. gordonii: The integration vector, pVMB20, was constructed to allow insertion of heterologous DNA sequences into the emm-6.1 gene present on the chromosome of GP232. pVMB20 is a novel Escherichia coli plasmid that does not replicate in Streptococcus and carries emm-6.1 and the erythromycin resistance marker ermC. The vector pVMB20 has been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, pursuant to the terms of the Budapest Treaty, and assigned Accession No. PTA433. The host-vector system GP232-pVMB20 is shown in detail in FIGS. 4 and 5. Specifically, the novel plasmid pVMB20 and novel bacteria S. gordonii GP246 were prepared by the following procedure.

Host-vector system for heterologous gene expression: [A] In the chromosome of the novel host strain S. gordonii GP232, a copy of the M6 protein gene (emm-6.1)(37), promoterless but with its own ribosomal binding site was integrated downstream of a strong chromosomal promoter. Adjacent to emm-6.1 is found ermC (34), whose coding sequence is interrupted by insertion in its BcII site of the 1.8-kb MboI fragment of pC221 containing a cat gene (38). GP232 expresses M6 on its surface and is resistant to chloramphenicol and sensitive to erythromycin. It was obtained using transformation to integrate heterologous DNA into the streptococcal chromosome. The structure is shown in the figure. The size (5.2-kb) of the heterologous DNA integrated into the chromosome in GP232 was determined by Southern blot analysis. The integration vector pVMB20, is a 6.3-kb E. coli plasmid that does not replicate in Streptococcus. It was obtained by subcloning in pBLUESCRIPT (Stratagene, La Jolla, Calif.) a 3.4-kb ClaI fragment of plasmid pVMB3 containing emm-6.1 and ermC by the procedure explained below. This is the same ClaI fragment which is integrated into the chromosome of GP232, the only difference being that in GP232 ermC is interrupted by cat. When pVMB20 is used as donor DNA in transformation of competent cells of S. gordonii GP232, erythromycin-resistant transformants are obtained by recombination between the integration vector and the homologous chromosomal sequences. The DNA fragment containing the cat gene is deleted in the chromosome of these transformants, whereas an intact ermC gene is restored. (FIG. 5)

(B) The E7 protein gene of HPV16(39) was cloned into the emm-6.1 sequence of pVMB20 to yield pVMB21. pVMB20 was digested with KpnI and HindIII and ligated with a KpnI/HindIII segment containing the E7 sequences obtained by in vitro DNA amplification (polymerase chain reaction) performed on plasmid pMBS21L/E7 (33). Amplification primers were designed in order to obtain "in frame" insertion of the 294 bp encoding for E7 into emm-6.1. Nucleotide sequence analysis of pVMB21 confirmed the expected structure of the M6:E7 translational fusion. pVMB21 was linearized and used to transform GP232. E7 was found to be expressed in 6% of the erythromycin-resistant transformants. In these transformants integration of the pVMB21 sequences produced a deletion involving the cat gene. The structure of GP246, a representative transformant, was confirmed by Southern blot analysis. The nucleotide sequence of the junction fragments of the M6:E7 gene fusion present on the chromosome of GP246 was also determined after cloning in pBLUESCRIPT the ClaI fragment containing the M6:E7 fusion.

Surface expression of the E7 protein: Expression of the E7 protein of HPV16 in S. gordonii on the surface of strain GP246 was verified by immunofluorescence using antibodies specific for either the M6 protein carrier or the E7 insert. GP246, containing the M6:E7 gene fusion exhibited positive fluorescence when reacted with either M6-specific or E7-specific polyclonal antibodies (FIG. 6), confirming the surface location of the E7 molecule and the M protein on the surface of S. gordonii. No fluorescence was observed when GP246 was reacted with known monoclonal antibody 10All, which is specific for an epitope of M6 whose coding region was contained in the KpnI/HindIII fragment deleted in the construction of M6:E7 gene fusion (FIG. 6).

To demonstrate that the E7-expressing recombinant streptococci in fact produce an M6:E7 fusion protein, S. gordonii cell extracts were analyzed by Western blot (FIG. 7). In cell extracts of GP246, the same bands reacted with E7- and M6-specific antibodies, whereas no E7-specific reactivity was found in the recipient GP232, whose extracts showed M6-specific reactivity (FIG. 7).

Substantially the same procedures used to construct M6:E7 a hybrid protein containing the C-terminal region of the M6 molecule and allergen-5 was successfully expressed on the S. gordonii. Western blots of the cell wall extract using allergen-5 specific antibodies established (as with M6:E7) that allergen-5 was indeed expressed in the cell wall fraction of the gordonii. Allergen 5 was obtained as described by Fang, et al. (66).

Immune response to fusion protein on streptococcal surface: The immunogenicity of the M6:E7 fusion protein was examined by immunizing mice with recombinant S. gordonii GP246 expressing the M6:E7 fusion protein. Control mice were immunized with the isogenic strain GP232 which express M6 protein. Sera from three animals immunized with each strain were pooled and tested by Western blot for their reactivity with purified E7 protein produced in Schizosaccharomyces pombe. FIG. 8 shows that animals immunized with strain GP246 containing surface M6:E7 produced antibodies reactive with the E7 protein, indicating that E7 protein is immunogenic when expressed as a fusion protein on the streptococcal surface. No antibodies to the E7 protein were seen in the sera of mice immunized with GP232 containing only M6.

The procedure which has been described permits the production of the non-pathogenic bacteria S. gordonii GP246 or other transformed gram positive bacteria which express a hybrid surface protein such as the novel hybrid surface antigen M6:E7. This novel antigen is a hybrid protein, the carboxy terminus of which is attached to the bacteria and is substantially the same C-terminal region normally found in other gram-positive surface proteins as explained above. The active polypeptide of this hybrid surface protein is the antigen E7 of HPV 16. When the bacteria is administered, e.g. by colonization, to an animal host in need of protection, the active polypeptide will elicit both humoral and cell mediated responses resulting in the production of a protective immune response against infection by papillomavirus.

It will be noted that the heterologous antigen of the hybrid surface protein of this invention prepared as described above is a viral antigen produced during viral replication. Thus, the process of this invention makes possible the production of novel gram positive bacteria for the delivery of viral antigens to an animal in sufficient quantities to elicit a protective immunogenic response to infection by a virus.

The novel M6:E7 and novel intermediate products of this invention are produced from two starting materials. These are pVV3:M6 and GP69. They can be prepared by the procedures described in the cited references. Therefore the novel products can all be obtained from known materials utilizing the procedures described herein. However, to assist in the practice of the invention and without admitting any necessity to do so pVV3:M6 and GP69 have been deposited at the American Type Culture Collection under the accession numbers ATCC 68003 and ATCC 68929, respectively.

The foregoing M6:E7 example illustrates the utilization of a non-pathogenic commensal gram positive bacteria normally present in the mammalian oral cavity to protect the whole body against viral infection. Analogously prepared non-pathogenic bacteria can be used to express and deliver other useful antigens by the processes described are illustrated herein. For example, protein antigens on the surface of mammalian tumors can be fused with the cell wall associated region of a surface antigen of any gram positive bacteria, the fusion gene inserted in a gram positive commensal, and the resulting bacteria employed as a vaccine to generate tumor specific immune responses useful in tumor therapy.

These examples illustrates only one aspect of the invention. The invention, in fact, provides a delivery system for any kind of polypeptide which may be useful to raise an immune response in an animal or for other useful purposes.

When utilized as a vaccine, the selected transformed commensal will be employed to colonize a mammal. It will produce the selected hybrid surface protein, the active polypeptide segment of which will elicit the production of protective antibodies.

Gram positive non-pathogenic bacteria normally found on the vaginal mucosa may be employed as contraceptives. For this utility, surface antigens of male sperm can be employed as the active polypeptide on the hybrid surface protein of the bacteria Lactobacillus acidophilus normally found on the surface of the vaginal or uterine