| PATENT ASSIGNEE'S COUNTRY | Belgium |
| UPDATE | 03.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 21.03.00 |
| PATENT TITLE |
Vaccine |
| PATENT ABSTRACT |
The Bordetella pertussis toxin is genetically modified to express a toxin protein which is deficient in target-cell receptor binding and is used in a vaccine for protection against whooping cough. -------------------------------------------------------------------------------- |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 01.07.98 |
| PATENT REFERENCES CITED |
Bowie, et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions", (1990), Science, 247, pp. 1306-1310. Pizza et al., "Bacterial Toxins Protein", (1990), Rappuoli et al. (Eds.), pp. 507-518, Gustav Fischer, Stuttgart, NY. Schmidt et al. (1989) Infect Immun., 57: 438-445. Burnette et al. (1988), Biotechnology, 6: 699-706. Ui, M., "The Multiple Biological Activated Pertussis Toxin, in Patho Genesis and Immunity in Pertussis", (1988), Wardlaw et al. (Eds.), pp. 121-144, John Wiley and Sons. Black et al., (1987), Infection and Immunity, vol. 55, pp. 2465-2470. Kunkel et al., (1987), Methods in Enzymology, vol. 154, pp. 367-382. Armstrong et al. (1987), Infection and Immunity, vol. 55, pp. 1294-1299. Locht et al. (1986), Science, vol. 232, pp. 1258-1264. Locht et al. (1987), Infection and Immunity, vol. 55, pp. 2546-2553. Capiau et al. (1986), Febs Letter, vol. 204, pp. 336-340. Stibitz et al., (1986), Gene, 50: 133-140. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An isolated DNA molecule comprising a recombinant coding sequence encoding a Bordetella pertussis holotoxin comprising a modified B-oligomer comprising either or both of a modified dimer D1or D2 wherein said modified dimer comprises an unmodified S4 subunit and either a modified S2 subunit or a modified S3 subunit and said modified B-oligomer optionally comprises an unmodified S5 subunit and further optionally comprises an unmodified S1 subunit, wherein said holotoxin comprising a modified B-oligomer is specifically reactive with antisera capable of recognizing a wild-type Bordetella pertussis holotoxin having no modified subunits; wherein said modified S2 subunit comprises one or more amino acid sequence modifications selected from the group consisting of a deletion of one or more of Tyr.sup.102, Tyr.sup.103 and Asn.sup.105, an amino acid substitution of either or both of Tyr.sup.102 and Tyr.sup.103 and a substitution of a negatively-charged amino acid for Asn.sup.105 ; and wherein said modified S3 subunit comprises one or more amino acid sequence modifications selected from the group consisting of a deletion of one or more of Tyr.sup.102, Tyr.sup.103 and Lyr.sup.105, an amino acid substitution of either or both of Tyr.sup.102 and Tyr.sup.103, and a substitution of a negatively-charged amino acid for Lys.sup.105. 2. A Bordetella pertussis holotoxin comprising a modified B-oligomer comprising either or both of a modified dimer D1 or D2 wherein said modified dimer comprises an unmodified S4 subunit and either a modified S2 subunit or a modified S3 subunit and said modified B-oligomer optionally comprises an unmodified S5 subunit and further optionally comprises an unmodified S1 subunit, wherein said holotoxin comprising a modified B-oligomer is specifically reactive with antisera capable of recognizing a wild-type Bordetella pertussis holotoxin having no modified subunits; wherein said modified S2 subunit comprises one or more amino acid sequence modifications selected from the group consisting of a deletion of one or more of Tyr.sup.102, Tyr.sup.103 and Asn.sup.105, an amino acid substitution of either or both of Tyr.sup.102 and Tyr.sup.103 and a substitution of a negatively-charged amino acid for Asn.sup.105 ; and wherein said modified S3 subunit comprises one or more amino acid sequence modifications selected from the group consisting of a deletion of one or more of Tyr.sup.102, Tyr.sup.103 and Lyr.sup.105, an amino acid substitution of either or both of Tyr.sup.102 and Tyr.sup.103, and a substitution of a negatively-charged amino acid for Lys.sup.105. 3. The modified B-oligomer of claim 2 comprising two modified dimers D1 and lacking both a S1 subunit and a S5 subunit. 4. The modified B-oligomer of claim 2 comprising two modified dimers D2 and lacking a S1 subunit and a S5 subunit. 5. The modified B-oligomer of claim 2 comprising a modified dimer D1 and a modified dimer D2 and lacking a S1 subunit and a S5 subunit. 6. The modified B-oligomer of claim 2 comprising two modified dimers D1 and an unmodified S5 subunit and lacking a S1 subunit. 7. The modified B-oligomer of claim 2 comprising two modified dimers D2 and an unmodified S5 subunit and lacking a S1 subunit. 8. The modified B-oligomer of claim 2 comprising a modified dimer D1 and a modified dimer D2, and an unmodified S5 subunit and lacking a S1 subunit. 9. The modified holotoxin of claim 2 comprising two modified dimers D1, an unmodified S5 subunit, and an unmodified S1 subunit. 10. The modified holotoxin of claim 2 comprising two modified dimers D2, an unmodified S5 subunit, and an unmodified S1 subunit. 11. The modified holotoxin of claim 2 comprising a modified dimer D1, a modified dimer D2, and unmodified S5 subunit, and an unmodified S1 subunit. 12. A recombinant plasmid which comprises the DNA molecule of claim 1 operatively linked to a regulatory region. 13. A host cell transformed with the recombinant plasmid of claim 12, wherein said host cell is selected from the group consisting of a yeast cell, an insect cell, a mammalian cell, an E. coli cell, a Streptomyces cell, a Bacillus cell, and a Salmonella cell. 14. The host cell of claim 13 which is E. coli. 15. A transformed host cell comprising the recombinant DNA molecule of claim 1 integrated into the genome of the host cell, wherein said host cell is selected from the group consisting of a yeast cell, an insect cell, a mammalian cell, an E. coli cell, a Streptomyces cell, a Bacillus cell, and a Salmonella cell. 16. A transformed host cell comprising the recombinant DNA molecule of claim 1 integrated into the genome of the host cell wherein said host cell is a Bordetella pertussis cell and wherein the genomic integration of said recombinant DNA molecule results in the expression of an inactive pertussis toxin. 17. A process or preparing a Bordetella pertussis holotoxin or portion thereof encoded by the DNA molecule of claim 1 which comprises: a) transforming a host cell selected from the group consisting of a yeast cell, an insect cell, a mammalian cell, an E. coli cell, a Streptomyces cell, a Bacillus cell, and a Salmonella cell with the recombinant DNA molecule of claim 1, and b) growing said transformed host cell in a suitable culture medium. 18. A whole cell vaccine for stimulating protection against whooping cough wherein such vaccine comprises an immunoprotective and non-toxic quantity of inactivated host cells of claim 16. 19. A vaccine or stimulating protection against whooping cough wherein such vaccine comprises an immunoprotective and non-toxic quantity of the Bordetella pertussis holotoxin or portion thereof of any of the preceding claims. 20. A method for protecting a human against disease symptoms associated with whooping cough infect on which comprises administering to such a human a safe and effective amount of the vaccine of any of the preceding claims. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to genetic modifications of the Bordetella pertussis toxin and to a vaccine comprising an immunoprotective amount of such protein. BACKGROUND OF THE INVENTION The members of the genus Bordetella are pathogenic microorganisms involved in the infection of the respiratory tract. The genus is comprised of four species; B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium. The most virulent species to man is B. pertussis, which is the etiologic agent of whooping cough. Current conventional pertussis vaccines contain whole but inactivated B. pertussis cells. Such cells are inactivated by treatment at 56.degree. C. for 30 minutes and/or treatment with formaldehyde. In spite of inactivation, such whole cell vaccines retain a substantial amount of toxicity. As a result, alternate pertussis vaccines are available which are prepared from avirulent or toxin-deficient strains of B. pertussis. However, these vaccines have proven to be much less protective than those prepared from virulent strains. See, for example, Wardlaw et al., J Med Microbiol 9:89-100 (1976). B. pertussis produces a number of toxins (pertussis toxin, adenylate cyclase, dermonecrotic toxin, and trachael cytotoxin) which destroy the clearance mechanisms of the respiratory tract, or interfere with the immune response (F. Mooi, Antonie van Leeuwenhoek, 54:465-474 (1988)). A wide variety of biological activities, such as histamine sensitization, insulin secretion, lymphocytosis promotion and immunopotentiating effects can be attributed to the pertussis toxin (J. Munoz, in Pertussis Toxin, p. 1-18, Sekura et al., Eds., Academic Press, New York, 1985). In addition, it has been shown that the administration of the B. pertussis toxin in mice protected them against subsequent challenge (Munoz et al., Infect Immun 32:243-250 (1981)). Pertussis toxin is therefore an important constituent in a vaccine against whooping cough and is included in the acellular component vaccines being tested and used in several countries (Sato et al., Lancet, 1984-I:122 (1984)). Paradoxically, the pertussis toxin, which is capable of eliciting an immune response, may itself be responsible for the harmful side effects associated with current vaccines (Steinman et al., Proc. Natl Acad Sci USA, 82:8733 (1985)). These harmful effects can range from simple flushing to permanent neurological damage and in some instances, death. The pertussis toxin is composed of five different subunits, designated S1 to S5 based on their electrophoretic migration in SDS-polyacrylamide gels. The subunits associate in the molar ratio of 1:1:1:2:1, respectively, to form the holotoxin. Functionally, the pertussis toxin can be divided into the A protomer, or S1 subunit, which contains adenosine diphosphate (ADP)-ribosylation activity, and the B-oligomer, comprised of subunits S2 through S5, which contains target cell receptor binding activities. Thus the B-oligomer is essential in bringing the A protomer into contact with the target-cell's membrane. Locht et al., Science 232: 1258-64 (1986), disclose that the subunits of the pertussis toxin are encoded by closely linked cistrons. Locht et al. further disclose the nucleotide sequence of the B. pertussis toxin gene and the amino acid sequences for the individual subunits. Locht et al., NAR 14:3251-61 (1986), reveal the cloning of a 4.5 kb DNA fragment from the B. pertussis toxin gene containing at least the S4 subunit and a portion of another subunit gene. Sequence analysis revealed that the mature S4 subunit is derived by proteolytic cleavage of a precursor molecule. Nicosia et al., Infect Immun 55:963-7 (1987), disclose expression of each of the five B. pertussis toxin subunits as fusions to DNA polymerase MS2. Antisera raised to these proteins were found not to be immunoprotective in vivo or in vitro. Locht et al., Infect Immun 55:2546-2553 (1987), disclose the expression of the S1 and S2 subunits of pertussis toxin in E. coli as fusions to 6 amino acid residues of beta-gacactosidase followed by 5 amino acids encoded by a polylinker. It was disclosed that the recombinant S1 subunit displayed enzymatic activities. A truncated version of the S1 subunit was disclosed in which the last 48 amino acid residues, i.e., the carboxy terminus, was deleted. Sclavo SpA, EP-A-232,229, published Aug. 12, 1987, disclose the cloning and expression of a B. Pertussis toxin gene, which contains subunits S1 through S5 in E. coli. Bellini et al., EP-A-281,530, published Sep. 7, 1988, disclose expression of mature B. pertussis subunits in B. subtilis Burnette et al., EP-A-306,318, published Mar. 8, 1989, report the subcloning and expression of individual B. pertussis toxin subunits in E. coli. Burnette et al. disclose that the S4 subunit could only be expressed upon removal of the signal peptide coding sequence. Burnette et al. also disclose S1 subunit analogs expressed in E. coli with modifications between amino acids Val.sup.7 to Pro.sup.14. Burns et al., U.S. Pat. No. 4,845,036, disclose a method for isolating the wild-type B. pertussis B-oligomer (i.e., subunits S2-S5) by dissociation of the holotoxin (i.e., subunits S1-S5). Sato et al., EP-A-296,765, published Dec. 28, 1988, disclose B. pertussis variants which produce mutant pertussis toxin proteins. The variants arose from exposure of virulent B. pertussis with nitrosoguanidine, a known mutagen. M. Ui, (in Pathogenesis and Immunity in Pertussis, Wardlaw et al., eds., p.121-145, Wiley & Sons, Chichester, 1988) discloses that certain chemical modifications, e.g., acylation, of the pertussis toxin lysine residues eliminate all biological activity. Methylation of the pertussis toxin, which also modifies lysine residues, does not affect the ADP-ribosylation activity but does reduce or abolish certain biological activities associated with the B-oligomer, for example, mitogenic activity, stimulation of glucose oxidation, promotion of lymphocytosis and histamine-sensitizing activity. Ui further discloses that methylation of dimer D2 (i.e., pertussis toxin subunits S3-S4), but not dimer D1 (i.e., pertussis toxin subunits S2-S4) or subunit S5, eliminates the mitogenic activity associated with the B-oligomer. There is no disclosure or suggestion, however, as to which specific regions or specific lysine residues of the B-oligomer are involved in the methylation or acylation. Hausman et al., Infect Immun 57:1760-64 (1989), disclose immunization of mice with the pertussis toxin dimeric subunits, D1 (i.e., S2-S4) and D2 (i.e., S3-S4). The antisera raised to these dimers were able to recognize B. pertussis toxin and neutralize its toxic effects in vitro. Capiau et al., U.S. Patent application Ser. No. 07/222,991,*filed Jul. 22, 1988 disclose modification of the B. pertussis toxin S1 subunit at amino acid position 26 (i.e., tryptophan). This residue can be modified either chemically or by site-directed mutagenesis to substantially inactivate the enzymatic activity of the S1 subunit. Bellini et al., Gene, 69:325-330 (1988), recite a general method for site-directed mutagenesis for double-stranded plasmid DNA. Bellini et al. disclose that their method is particularly valuable where long deletions are needed. Exemplified is the deletion of the B. pertussis S2 subunit signal sequence coding region located on an E. coli--B. subtilis shuttle vector. Black et al., EP-A-275, 689, published Jul., 27, 1988 and Infect Immun 55:2465-70 (1987), disclose expression of the S4 subunit in E. coli. In addition, Black et al. disclose mutations in the B. pertussis toxin gene that were either deletions generated by Bal31 exonuclease or insertions with the kanamycin resistance gene. These mutations were then introduced by allelic exchange into the B. pertussis chromosome. Klein et al., EP-A-322,115, published Jun. 28, 1989, disclose substitution mutations of the B. pertussis toxin. Klein et al. also disclose deletion mutations of the S1 subunit. However, of the deletion mutations disclosed, only one mutation, Glu.sup.129, was weakly reactive against antibodies to the S1 subunit. It is an object of this invention to provide an improved B. pertussis vaccine comprising a modified B. pertussis toxin or subunits thereof which are immunogenic, yet non-toxic. SUMMARY OF INVENTION The present invention relates to a recombinant DNA molecule which encodes a protein specifically reactive with antibodies against the wild-type pertussis toxin but which is defective in pertussis toxin target cell receptor binding. In related aspects, this invention is a recombinant plasmid which comprises the recombinant DNA molecule of this invention operatively linked to a regulatory region. Said regulatory region contains regulatory sequences necessary for transcription of the protein coding sequence and subsequent translation in a host cell transformed with the recombinant plasmid of the invention. This invention also relates to a pertussis toxin protein encoded by the recombinant DNA molecule of the invention, which is specifically reactive with antibodies against the wild-type pertussis toxin but which is also defective in pertussis toxin target cell receptor binding. In another aspect, this invention is a vaccine for stimulating protection against whooping cough wherein such vaccine comprises an immunoprotective and non-toxic quantity of the protein encoded by the recombinant DNA molecule of the invention. For vaccinal purposes, the protein of the invention may be purified away from the host cell or cell culture medium, or alternatively, it may be associated with the outer surface membrane of the host cell. This invention further relates to a process for preparing the protein of the invention. This process comprises growing a host cell transformed with the recombinant DNA molecule of this invention in a suitable culture medium. DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery that modification of one or more of the naturally occurring amino acid residues of the Bordetella pertussis toxin B-oligomer DNA coding sequence greatly reduces the binding of pertussis toxin to target cell receptors thereby greatly reducing the toxicity of the pertussis toxin while retaining its ability to elicit a protective immunogenic response. Many, but not all of the biological activities of the pertussis toxin (PT) are the result of three essential molecular steps. The first step involves the binding of PT to the receptors on the target cell membranes via the B-oligomer (subunits S2 through S5). Next, the S1 subunit must translocate into the cytoplasm of the target cell. Finally, the internalized S1 subunit has to express its enzymatic activity which includes NAD-glycohydrolysis and ADP-ribosylation. (For a review, see Ui, M., in Pathogenesis and Immunity in Pertussis, p. 121-145, Wardlaw and Parton, eds., Wiley and Sons, Chichester, 1988). Elimination of any of these three steps drastically diminishes the biological activities of said toxin. Therefore, a modified pertussis toxin which is incapable of binding to target cell receptors, drastically reduces or eliminates the toxic activities associated with the pertussis toxin. The B-oligomer, which is associated with adherence or binding of PT to host cells, is composed of subunits S2-S5. The B-oligomer can be further divided into two dimers, D1 and D2, which are connected by subunit S5. Dimer D1 comprises subunits S2 and S4, whereas dimer D2 comprises subunits S3 and S4 (see, Tamura et al., Biochemistry, 21:5516-22 (1982)). Each dimer specifically interacts with different target cell receptor molecules. For example, D1 appears to be responsible for the binding of PT to such glycoproteins as haptoglobin or fetuin (see, Francotte et al., in Vaccine 89, p. 243-247, Lerner et al., eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), whereas D2 can specifically bind to Chinese Hamster Ovary (CHO) cell membranes (see, Brennan et al., J Biol Chem, 263:4895-99 (1988)). It is presumed that this binding specificity is attributed to the structural differences between S2 and S3. The nucleotide sequence for the Bordetella pertussis toxin gene is disclosed by Locht et al. (Science, 232:1258-64 (1986)): Chemical modification of the pertussis toxin can affect its biological activities. Acylation, for example, eliminates all biological activity associated with the pertussis toxin due to disruption of the quaternary structure (see, Nogimori et al., Biochim Biophys Acta, 801:220-231 (1984)). Methylation of the pertussis toxin, which modifies lysine residues, does not affect the ADP-ribosylation activity but does reduce or abolish activity associated with the B-oligomer (see Ui, M., in Pathogenesis and Immunity in Pertussis, p. 121-145, Wardlaw and Parton, eds., Wiley and Sons, Chichester, 1988). Furthermore, Ui discloses that methylation of dimer D2, but not dimer D1 or subunit S5, eliminates the mitogenic activity associated with the B-oligomer. Therefore, Ui concludes that the lysine residues play a role in the attachment of D2 to the host cell's surface, but not the attachment of D1. It has also been shown that iodination of pertussis toxin severely reduces some biological activities, such as hemagglutination and CHO cell clustering. See, Armstrong et al., Infect Immun, 55:1294-99 (1987). Armstrong et al. also disclose a method to radioiodinate the wild-type pertussis toxin in the presence of fetuin-agarose and still retain biological activity. It was reported that by using such method, all of the PT subunits were iodinated. However, Armstrong et al. report that the mechanism for the observed reduction in biological activity is not known. The present invention thus describes a B. pertussis toxin DNA sequence which encodes a protein which is defective in target cell receptor binding activity and preferably lacks S1 subunit enzymatic activity as well, yet retains the capability to be recognized by anti-Pertussis Toxin antibodies. Target cell receptor binding activity can be assayed by haptoglobin-binding as described by Francotte et al. (in Vaccine 89, p.243-247, Lerner et al., eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) or by CHO cell binding/cytotoxicity assays as described by Brennan et al. (J Biol Chem, 263:4895-99 (1988)) or Burns et al. (Infect Immun, 55:24 (1987)). S1 subunit enzymatic activity can be measured by the ADP-ribosylation assay as described by Burnette et al. (Science, 242:72 (1988)). DNA molecules comprising the recombinant DNA molecule of this invention can be derived from any B. pertussis strain using known techniques, e.g., isolating the gene from a gene bank, making complementary or cDNAs from a mRNA template or via the polymerase chain reaction (see, U.S. Pat. No. 4,800,159) or from isolates of clinical specimens. Alternatively, such recombinant DNA molecule may be synthesized by standard DNA synthesis techniques. Furthermore, various B. pertussis strains are publicly available from commercial depositories, e.g., from the American Type Culture Collection (ATCC), Rockville, Md., U.S.A. As used herein, the term "DNA sequence which encodes a protein specifically reactive with antibodies against wild-type pertussis toxin but which is defective in pertussis toxin target cell receptor binding" means a DNA coding sequence which encodes a protein with decreased target cell binding activity relative to the wild-type pertussis toxin but still retains the ability to be recognized by anti-pertussis toxin antibodies, such as a coding sequence which encodes a protein comprising all the subunits (S1, S2, S3, S4 and S5) substantially as described by Locht et al. (Science 232:1258-64 (1986)) and all mutations or mutants thereof. The terms "mutations" or "mutants" as applied to the B oligomer or S2 or S3 subunits encompass any derivative of the recombinant DNA molecule of this invention which encodes a protein which is defective in target cell receptor binding and still retains the ability to be recognized by anti-Pertussis Toxin antibodies. Such mutations can be prepared by the deletion, addition, substitution, or rearrangement of amino acids and their nucleic acid coding sequences, or alternatively, by chemical modifications thereof. Preferred embodiments of the recombinant DNA molecule of this invention include, but are not limited to, a recombinant DNA molecule containing amino acid deletions in Asn.sup.105, or Tyr.sup.102, or Tyr.sup.102-103 of subunit S2, and/or Lys.sup.10, or Tyr.sup.92, or Lys.sup.93, or Lys.sup.105, or Tyr.sup.102, or Tyr.sup.102-103 of subunit S3, or single amino acid substitutions, e.g., Asn.sup.105 to Asp of subunit S2. The most preferred embodiments include, but are not limited to, deletions of Asn.sup.105, or Tyr.sup.102, or Tyr.sup.102-103 in subunit S2 and/or Lys.sup.105, or Tyr.sup.102, or Tyr.sup.102-103 in subunit S3. Mutations in S1 (e.g., deletions of Trp.sup.26, or His.sup.35, or Ser.sup.40, or Glu.sup.129) are disclosed in U.S. application Ser. No. 07/381,888,* filed Jul. 18, 1989, the entire disclosure of which is hereby incorporated by reference herein. Mutations of other S1 subunit amino acid residues (i.e., substitutions of Arg.sup.9, Arg.sup.13, Glu.sup.129) are disclosed by Pizza et al., Science, 246:497-500 (1989) and EP-A-O 396 964, and also in EP-A-0306318. These references are incorporated by reference herein. Preferably the DNA coding sequence of this invention will encode a protein that resembles the natural or wild-type protein as much as possible in tertiary structure, i.e., a protein which is able to be recognized by anti-pertussis toxin antibodies, yet is deficient in target cell receptor binding activity. Other embodiments of the recombinant DNA molecule of this invention include a pertussis toxin coding sequence which encodes some, but not all of the subunits. Exemplary embodiments include, but are not limited to, D1 (S2 and S4), D2 (S3 and S4), S1-S2-S4, S1-S3-S4 and the B-oligomer (S2, S3, S4 and S5). In another embodiment, the recombinant DNA molecule of the invention can be in the form of a hybrid, that is, a coding sequence which encodes a fusion polypeptide containing additional sequences which can carry one or more epitopes from other PT subunits, for example, [S2 epitope]-[S3 subunit], etc., other B. pertussis antigens, or other non-B. pertussis antigens. Alternatively, the recombinant DNA molecule of the invention can be fused to the DNA coding sequence of a carrier polypeptide which has immunostimulating properties, as in the case of an adjuvant, or which otherwise enhances the immune response to the B. pertussis toxin subunits(s), or which is useful in expressing, purifying or formulating the B. pertussis toxin subunits(s). The recombinant DNA molecule of this invention may comprise additional DNA sequences, including e.g., a regulatory element, one or more selectable markers, and sequences that code for replication and maintenance functions. The regulatory region typically contains a promoter found upstream from the coding sequence of this invention, which functions in the binding of RNA polymerase and in the initiation of RNA transcription. In other words, the regulatory element or region is operatively linked to the coding sequence of this invention. It will be appreciated by one of skill in the art that the selection of regulatory regions will depend upon the host cell employed. This invention also relates to a recombinant DNA plasmid comprising the recombinant DNA molecule of this invention. Another aspect of this invention is a host cell transformed with the recombinant DNA molecule of this invention. Such host cell is capable of growth in a suitable culture medium and expressing the coding sequence of the invention. Such host cell is prepared by the method of this invention, i.e., by transforming a desired host cell with the plasmid of this invention. Such transformation is accomplished by utilization of conventional transformation techniques. Moreover, the recombinant DNA molecule of this invention can be integrated into the host cell's genome by conventional techniques, e.g., homologous recombination. The most preferred host cells of this invention include those belonging to the species E. coli and the genus Bordetella. Other host cells which may be suitable include, but are not limited to, mammalian cells, insect cells, yeast and other bacterial cells, e.g., Streptomyces, Bacillus and Salmomella. Thus, this invention and the product thereof is not limited to any specific host cell. The present invention also relates to a protein encoded by the recombinant DNA molecule of this invention. Preferably such protein is produced by the transformed host cell of this invention, but such protein may be prepared by conventional peptide synthesis techniques. The protein of this invention preferably has no more than about 50% of the haptoglobin binding or CHO cytotoxicity as compared to wild-type pertussis toxin. Most preferably, the protein of this invention has less than 10% and more preferably less than 5% of either of the assayed activities as compared to wild-type pertussis toxin. The present invention also relates to a method of producing the protein encoded by the recombinant DNA molecule of this invention which comprises culturing the transformed host of the invention in an appropriate culture media and the isolation of such protein. By "appropriate culture media" is meant that media which will enable such host to express the coding sequence of the invention in recoverable quantity. It will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the protein so produced is accomplished from a culture lysate of the host, or if appropriate, directly from the host's culture medium, and such isolation is carried out by conventional protein isolation techniques. See, for example, Burns et al., U.S. Pat. No. 4,845,036. In a preferred embodiment of this invention, the coding sequence of the protein of the invention is expressed in a transformed B. pertussis host cell to produce an immunogenic yet substantially inactivated protein, i.e., a protein that is deficient in target-cell receptor binding, and in addition, may optionally be deficient in ADP- ribosyltransferase activity, but which is still specifically recognized by anti-Pertussis Toxin antibodies. In such a system, sequences that encode B. pertussis toxins are typically located on a suicide vector. Such suicide vector contains a sufficient amount of bacterial DNA to propagate the suicide vector in E. coli or some other suitable host. Such suicide vector also contains a sufficient amount of B. pertussis DNA flanking the toxin subunit coding sequence so as to permit recombination between a B. pertussis host deficient in the toxin gene and the heterologous toxin gene. It is understood to one skilled in the art that it is not essential to use a B. pertussis host deficient in the toxin gene, but that the absence of the toxin gene in the host prior to recombination will facilitate the screening and isolation of recombinant hosts which have incorporated the gene of interest. The recombinant B. pertussis arising from such homologous recombination are then selected by standard techniques. See, e.g., Stibitz et al., Gene 50:133-140 (1986). The invention also encompasses a vaccine capable of inducing immunity against whooping cough. Such vaccine comprises an immunoprotective and non-toxic amount of the protein of the invention. Such vaccine typically contains 1-500 .mu.g, preferably 5-25 .mu.g of the protein of this invention, but is not limited to use of these amounts. Further embodiment of the present invention include a whole cell vaccine for stimulating protection against whooping cough. Such vaccine comprises the protein of this invention expressed on the surface of transformed host cells of this invention. The transformed host cells are subsequently inactivated by conventional techniques to constitute an immunoprotective and non-toxic vaccine. Other antigens which are known to be desirably administered in conjugation with pertussis toxin may also be included in the vaccine of this invention. Such additional components are known to those skilled in the art. Preferably additional components include tetanus toxoid and/or diptheria toxoid as well as filamentous hemagglutinin (FHA). agglutinogens 2 and 3, the 69 kD antigen, and/or any other protective antigen of B. pertussis. The provision of such a vaccine thus allows for another aspect of the present invention, i.e., a method for immunizing a human against whooping cough which comprises administering the vaccine of the subject invention to such human. The mode of administration of the vaccine of the invention may be any suitable route which delivers an immunoprotective amount of the protein of the subject invention to the host. However, the vaccine is preferably administered parenterally via the intramuscular or subcutaneous routes. Other modes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally or intravenously. The vaccine of the invention may be prepared as a pharamaceutical composition containing an immunoprotective, non-toxic and sterile pharmaceutically acceptable carrier. In the vaccine of the invention, an aqueous solution of the protein of this invention can be used directly. Alternatively, the protein of this invention, with or without prior lyophilization, can be mixed together or with any of the various known adjuvants. Where the administration of the vaccine is parenteral, the protein of the invention can be optionally admixed or absorbed with any conventional adjuvant to enhance an immune response. Such adjuvants include among others, aluminum hydroxide, aluminum phosphate, muramyl dipeptide and saponins, such as Quil A. As a further exemplary alternative of the preparation of the vaccine of the invention, the protein of the invention can be encapsulated within microparticles such as liposomes. In yet another exemplary alternative of the preparation of the vaccine of the invention, the protein of the invention can be administered with an immunostimulating macromolecule, for example, tetanus toxoid. Alternatively, an aqueous suspension or solution containing the protein of the invention preferably is buffered at physiological pH. The protein of the invention may also be designed for oral digestion. Vaccine preparation is generally described in New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md., 1978. Encapsulation within liposomes is described by Fullerton, U.S. Pat. No. 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Pat. No. 4,372,945 and Armor et al., U.S. Pat. No. 4,474,757. Use of Quil A is disclosed by Dalsgaard et al., Acta Vet Scand 18:349 (1977). It is preferred that the vaccine of the invention, when in a pharmaceutical preparation, be present in unit dosage forms. The appropriate prophylactically effective dose can be determined readily by those of skill in the art. The effective amount of protein contained in the vaccine of this invention may be in the range of effective amounts of antigen in conventional B. pertussis acellular or component vaccines, i.e., 5-25 .mu.g of protein per unit dose. This dose may optionally be delivered with various amounts of filamentous hemagglutin (FHA) (approximately 10-25 .mu.g per dose) and/or agglutinogens or other antigens. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the age, general health, the time of administration, the route of administration, synergistic effects with any other drugs being administered, and the degree of protection being sought. The administration can be repeated at suitable intervals if necessary. The examples which follow are illustrative but not limiting of the present invention. Restriction enzymes and other reagents were used substantially in accordance with the vendors instructions. |
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