WO1992021374A1

WO1992021374A1 - Induction of immune response against tetanus toxin through administration of mycobacteria expressing tetanus toxin or fragments

Info

Publication number: WO1992021374A1
Application number: PCT/US1992/005023
Authority: WO
Inventors: Charles K. Stover; Vidal Dela Cruz
Original assignee: Medimmune, Inc.
Priority date: 1991-06-06
Filing date: 1992-06-01
Publication date: 1992-12-10
Also published as: EP0587765A4; AU2221992A; EP0587765A1; CA2110687A1

Abstract

A method of inducing an immune response to a tetanus toxin in an animal which comprises administering to the animal mycobacteria transformed with at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin or a fragment or derivative thereof. In one embodiment, the immune response to tetanus toxin is induced by administering to an animal a vaccine including BCG which have been transformed with an expression vector DNA sequence which encodes Fragment C of tetanus toxin.

Description

INDUCTION OF IMMUNE RESPONSE AGAINST TETANUS TOXIN THROUGH ADMINISTRATION OF MYCOBACTERIA EXPRESSING TETANUS TOXIN OR FRAGMENTS

This invention relates to inducing an immune response against tetanus toxin. More particularly, this invention relates to inducing an immune response against tetanus toxin by administering to an animal mycobacteria transformed with DNA which encodes for a tetanus toxin or a fragment(s) or derivative(s) thereof.

Vaccination against tetanus is extremely effective in the prevention of this disease in many countries. In general, current tetanus vaccines are produced by formaldehyde treatment of tetanus toxin produced by the anaerobic bacterium C. tetani to produce the immunogenic toxoid. Impurities incorporated into tetanus vaccines during the formaldehyde treatment, however, may be partly responsible for adverse effects which are sometimes associated with vaccination against tetanus. (Bizzini, et al., Eur. J. Biochem., Vol. 39, pgs. 171-181 (1973)); Bizzini, "Tetanus", in Bacterial Vaccines, pgs. 37-67, Germanier, R. , ed. , Academic Press. (1984).

As an alternative to the formaldehyde treatment of tetanus toxin, DNA encoding Fragment C of tetanus toxin has been cloned into bacteria which then express such DNA. Halpern, et al., Infection and Immunity, Vol. 58, No. 4, pgs. 1004-1009 (April 1990) disclose the cloning of C. tetani DNA corresponding to Fragment C of tetanus toxin into an expression vector which is

SUBSTITUTE SHEET expressed in E.coli. Recombinant Fragment C expressed by E.coli was purified^" and administered to mice. Immunization of mice with such recombinant Fragment C resulted in the production of antibodies that were able to protect the mice against a challenge with tetanus toxin. Fairweather, et al., Infection and Immunity, Vol. 58, No. 5, pgs. 1323-1326 (May 1990) disclose the introduction of a plasmid, which expresses Fragment C of tetanus toxin into a Salmonella tvphimurium mutant. The transformed mutants are then administered to mice orally or intravenously. The oral or intravenous administration of the transformed mutants was able to induce protective immunity in mice against a lethal tetanus toxin challenge.

In accordance with an aspect of the present invention, there is provided a method of inducing an immune response in an animal against tetanus toxin. The method comprises administering to the animal mycobacteria transformed with DNA which includes at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment(s) or derivative(s) thereof. The mycobacteria are administered in an amount effective to induce an immune response in the animal to a tetanus toxin.

Ine one embodiment, the at least one DNA sequence may encode for tetanus toxin, which is a 150 kDa protein of 1,315 amino acids (Eisel, et al., EMBO J., Vol. 10, pgs. 2495-2502 (1986)), or fragment(s) or derivative(s) thereof. In one embodiment, the at least one DNA sequence encodes Fragment C of tetanus toxin or a fragment(s) or derivative(s) thereof. Fragment C is a 50 Da peptide which comprises the 451 C-terminal amino acids pf tetanus toxin. (Helting, et al., J.Biol. Chem., Vol 252, pgs. 187-193 (1977)).

Mycobacteria which may be transformed with the at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin or a fragment or derivative thereof include, but are not limited to, Mvcobacterium bovis-BCG,

SUBSTITUTESHEET M.smeqmatis- M.avium, M.phlei, M.fortuitum, M.lufu,

M.paratubereulosis. M.habana, M.scrof laceum, and

M.intracellulare. Preferably, the mycobacterium is M.bovis-BCG or a mutant thereof.

The at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment(s) or derivative(s) thereof, is contained within an expression vector which may be employed to transform the mycobacteria.

The expression vector may be, for example, a temperate shuttle phasmid or a bacterial mycobacterial shuttle plasmid. Each of these vectors may be used to introduce the at least one DNA sequence encoding a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, stably into mycobacteria, in which the at least one DNA sequence may be expressed. When a shuttle phasmid, which replicates as a plasmid in bacteria and a phage in mycobactreia, is employed, integration of the phasmid, which includes the at least one DNA sequence encoding a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof into the mycobacterial chromosome occurs through site-specific integration. The at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin, or fragment or derivative thereof, is replicated as part of the chromosomal DNA. When a bacterial-mycobacterial shuttle plasmid is employed, the at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin or fragment or derivative thereof is stably maintained extrachromosomally in a plasmid. Expression of the at least one DNA sequence occurs extrachromosomally (e.g., episomally). For example, the at least one DNA sequence is cloned into a shuttle plasmid, and the plasmid is introduced into a mycobacterium such as those hereinabove described, wherein the plasmid replicates episomally.

SSTITUTE SHEI Examples of such shuttle phasmids and bacterial-mycobacterial shuttle plasmids are further described in Application Serial No. 361,944, filed June 5, 1989, which is hereby incorporated by reference.

In accordance with one embodiment, the mycobaceteria are transformed with an expression vector which comprises at least one DNA sequence encoding a protein or polypeptide which elicits antibodies against tetanus toxin or a fragment or derivative thereof, and a promoter selected from the class consisting of mycobacterial promoters and mycobacteriophage promoters for controlling expression of the at least one DNA sequence encoding the protein or polypeptide which elicits antibodies against tetanus toxin or fragment or derivative thereof.

Mycobacterial and mycobacteriophage promoters which may be employed include, but are not limited to, mycobacterial promoters such as the BCG HSP60 and HSF70 promoters; the mycobactin promoter from M. tuberculosis and BCG; the mycobacterial 14 kda and 12 kda antigen promoters; the mycobacterial o-antigen promoter from M.tuberculosis or BCG; the MBP-70 promoter, the mycobacterial 45 kda antigen promoter from M.tuberculosis or BCG; the superoxide dismutase promoter; the mycobacterial asd promoter, and mycobacteriophage promoters such as the Bxbl, LI, L5, and TM4 promoters. In one embodiment, the promoter is a mycobacterial heat shock protein promoter such as HSP60 or HSP70. The promoter sequence may, in one embodiment, be part of an expression cassette which also includes a portion of the gene normally under the control of the promoter. For example, when a mycobacterial HSP60 or HSP70 promoter is employed, the expression cassette may, within the scope of the present invention, include, in addition to the promoter, a portion of the gene for the HSP60 or HSP70 protein. When the expression cassette and the DNA encoding the tetanus toxin or fragment or derivative thereof are expressed, the protein expressed by the cassette and the DNA encoding the protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, is a fusion protein of a fragment of a mycobacterial protein (eg. , the HSP60 or HSP70 protein), and the protein or polypeptide which elicits antibodies against tetanus toxin, or fragment or derivative thereof.

In a preferred embodiment, the transcription initiation site, the ribosomal binding site, and the start codon, which provides for the initiation of the translation of mRNA, are each of mycobacterial origin. The stop codon, which stops translation of mRNA, thereby terminating synthesis of the protein or polypeptide which elicits antibodies against tetanus toxin or a fragment or derivative thereof, and the transcription termination site, may be of mycobacterial origin, or of other bacterial origin, or such stop codon and transcription termination site may be those of the DNA encoding the protein or polypeptide which elicits antibodies against tetanus toxin or a fragment or derivative thereof.

Preferably, the mycobacterial promoter is a BCG promoter, and the mycobacterium is BCG.

The expression vector may additionally include DNA which encodes other proteins or polypeptides such as antigens, anti-tumor agents, enzymes, lymphokines, pharmacologic agents, immunopotentiatorβ, and reporter molecules of interest in a diagnostic context.

It is also contemplated that the expression vector may also include a or selectable marker.

Selectable markers which may be encoded include, but are not limited to, the β-galactosidase marker, the kanamycin resistance marker, the chloroamphenicol resistance marker, the neomycin resistance marker, and the hygromycin resistance marker.

In accordance with one embodiment, the vector further includes a mycobacterial origin of replication.

In accordance with another embodiment, the vector may be a plasmid. The plasmid may be a non-shuttle plasmid, or may be a shuttle plasmid which further includes a bacterial origin of replication such as an E.coli origin of replication, a Bacillus origin of replication, a Staphylococcus origin of replication, a Streptomvces origin of replication, or a pneumococcal origin of replication. In one embodiment, the shuttle plasmid includes an E. coli origin of replication.

In accordance with yet another embodiment, the vector may further include a multiple cloning site, and the DNA encoding the protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, is inserted in the multiple cloning site.

In addition to the DNA encoding a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, and the mycobacterial promoter for controlling expression of the DNA encoding the protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, the expression vector may, in one embodiment, further include a DNA sequence encoding bacteriophage integration into a mycobacterium chromosome. Bacteriophages from which the DNA sequence encoding bacteriophage integration into a mycobacterium chromosome may be derived include, but are not limited to, mycobacteriophages such as but not limited to, the L5, LI, Bxbl, and TM4 mycobacteriophages; the lambda phage of E. coli; the toxin phages of Corvnebacteria; phages of Actinomvcetes and Norcardi the *C31 phage of Streptomvces; and the P22 phage of Salmonella. Preferably, the DNA sequence encodes mycobacteriophage integration into a mycobacterium chromosome. The DNA sequence which encodes bacteriophage integration into a mycobacterium chromosome may include DNA which encodes integrase, which is a protein that provides for integration of the vector into the mycobacterial chromosome. Preferably, the DNA sequence encoding mycobacterial phage integration also includes DNA which encodes an attP site.

SUBSTITUTE SHΞ=T The DNA encoding the attP site and the integrase provides for an integration event which is referred to as site-specific integration. DNA containing the attP site and the integrase gene is capable of integrating into a corresponding attB site of a mycobacterium chromosome.

It is to be understood that the exact DNA sequence encoding the attP site may vary among different phages, and that the exact DNA sequence encoding the attB site may vary among different mycobacteria.

Examples of expression vectors including a mycobacterial promoter or a mycobacteriophage promoter are further described in application Serial No. 642,017 filed January 16, 1991, which is a continuation of application Serial No. 552,828, filed July 16, 1990, now abandoned. The contents of application Serial No. 642,017 are hereby incorporated by reference.

In another embodiment, the mycobacteria are transformed with a DNA which comprises a first DNA sequence which is a phage DNA portion encoding bacteriophage integration into a mycobacterium chromosome, and the at least one DNA sequence encoding a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof.

The term "phage DNA portion", as used herein, means that the DNA sequence is derived from a phage and lacks the DNA which is required for phage replication.

Bacteriophages from which the phage DNA portion may be derived include, but are not limited to, those hereinabove described. Preferably, the phage DNA portion encodes mycobacteriophage integration into a mycobacterium chromosome.

Preferably the first DNA sequence includes DNA encoding integrase and most preferably, the first DNA sequence also includes DNA which encodes an AttP site, thereby providing for site-specific integration as hereinabove described. DNA containing the AttP site and the integrase gene is capable of

SUBSTITUTE SHEET integration into a corresponding AttB site of a mycobacterium chromosome."""^"

The integration event results in the formation of two new junction sites called AttL and AttR, each of which contain part of each of AttP and AttB. The inserted and integrated non-phage DNA which includes the first and second DNA sequences, is flanked by the AttL and AttR sites. The insertion and integration of the phage DNA portion results in the formation of a transformed mycobacterium.

The DNA may also include a selectable marker or markers such as those hereinabove described, and may, if desired, include DNA encoding for other proteins or polypeptides such as antigens, anti-tumor agents, enzymes, lymphokines, pharmacologic agents, immunopotentiatorβ, and reporter molecules of interest in a diagnostic context.

The phage DNA portion of the present invention, which includes the first DNA sequence encoding mycobacterium phage integration into a mycobacterium chromosome, and the at least one DNA sequence encoding a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, may be constructed through genetic engineering techniques known to those skilled in the art. In a preferred embodiment, the phage DNA portion may be a plasmid including, in addition to the DNA encoding integration and the at least one DNA sequence encoding a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, an origin of replication for any of a wide variety of organisms, which includes, but is not limited to, E.coli, Streptomvces species, Bacillus species, Staphylococcus species, Shigella species, Salmonella species and various species of

SU_BSTITUTE SHEET pneumococci. Most preferably, the plasmid includes an origin of replicationTor E.coli.

The phage DNA portion also may include a suitable promoter. Suitable promoters include, but are not limited to, mycobacterial promoters such as the BCG HSP60 and HSP70 promoters; mycobactin promoters of M. tuberculosis and BCG, the superoxide dismutase promoter, the o- ntigen promoter of M. tuberculosis and BCG, the MBP-70 promoter, the 45 kda antigen promoter of M. tuberculosis and BCG; and the mycobacterial asd promoter; the mycobacterial 14 kda and 12 kda antigen promoters; mycobacteriophage promoters such as the Bxbl promoter, the LI and L5 promoters, and the TM4 promoters; E.coli promoters; or any other suitable promoter. The selection of a suitable promoter is deemed to be within the scope of those of ordinary skill in the art from the teachings contained herein.

The promoter sequence may, in one embodiment, be part of an expression cassette which also includes a portion of the gene normally under the control of the promoter, as hereinabove described. For example, when a mycobacterial HSP60 or HSP70 promoter is employed, the expression cassette may include, in addition to the promoter, a portion of the gene for the HSP60 or HSP70 protein. When the expression cassette and the DNA sequence encoding the protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, the protein expressed by the cassette and the DNA encoding the protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof, is a fusion protein of a fragment of a mycobacterial protein (eg., the HSP60 or HSP70 protein), and the protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof.

In one embodiment, the transcription initiation site, the ribosomal binding site, and the start codon, which provides for the initiation of the translation of mRNA, are each of mycobacterial origin, as hereinabove described. The stop codon,

_.UBSTITUTE SHEET which stops translation of mRNA, thereby terminating synthesis of the tetanus— oxin or fragment or derivative thereof, and the transcription termination site, may be of mycobacterial origin, or of other bacterial origin, or such stop codon and transcription termination site may be those of the DNA encoding the protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment or derivative thereof.

Examples of DNA which includes a first DNA seqeunce which is a phage DNA portion encoding bacteriophage integration into a mycobacterium are further described in application Serial No. 553,907, filed July 16, 1990, the contents of which are incorporated by reference.

The mycobacteria which are transformed with at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin or a fragment or derivative thereof, may be utilized to form a composition such as, for example, a vaccine, for inducing an immune response to a tetanus toxin in an animal. The animal may be a human or non-human animal. To form such a vaccine or therapeutic agent, the transformed mycobacteria are administered in conjunction with a suitable pharmaceutical carrier. As representative examples of suitable carriers there may be mentioned: mineral oil, alum, synthetic polymers, etc. Vehicles for vaccines are well known in the art and the selection of a suitable vehicle is deemed to be within the scope of those skilled in the art from the teachings contained herein. The selection of a suitable vehicle is also dependent upon the manner in which the vaccine is to be administered. The vaccine may be in the form of an injectable dose and may be administered intramuscularly, intravenously, orally, intradermally, or by subcutaneous administration.

Other means for administering the vaccine should be apparent to those skilled in the art from the teachings herein; accordingly, the scope of the invention is not to be limited to a particular delivery form.

SUBSTITUTESHEET When the transformed mycobacteria are employed as a vaccine, such a vaccine has important advantages over other presently available vaccines. Mycobacteria have, as hereinabove indicated, adjuvant properties among the best currently known and, therefore, stimulate a recipient's immune system to respond with great effectiveness. Also, mycobacteria may stimulate long-term memory or immunity. It thus may be possible to prime long-lasting T cell memory, which stimulates secondary antibody responses neutralizing to the tetanus toxin.

The invention will now be described with respect to the following examples; however, the scope of the present invention is not to be limited thereby.

Example 1 A. Construction of plasmids including mycobacterial promoter expression cassette.

1. Construction of PYUB125

Plasmid pAL5000, a plasmid which contains an origin of replication of M. fortuitum, and described in Labidi, et al., FEMS Microbiol. Lett.. Vol. 30, pgs. 221-225 (1985) and in Gene, Vol. 71, pgs. 315-321 (1988), is subjected to a partial Sau 3A digest, and 5kb fragments are gel purified. A 5kb fragment is then ligated to Bam HI digested pIJ666 (an. E. coli vector containing an E. coli origin of replication and also carries neomycin-kanamycin resistance, as described in Kieser, et al., Gene, Vol. 65, pgs. 83-91 (1988) to form plasmid pYUB12. A schematic of the formation of plasmid pYUB12. A schematic of the formation of plasmid pYUB12 is shown in Figure 1. pYUB12 and pIJ666 were then transformed into M. smeqmatis and BCG._λ Neomycin-resistant transformants that were only obtained by pYUB12 transformation confirmed that pAL5000 conferred autonomous replication to pIJ666 in M. smeqmatis and BCG.

Shotgun mutagenesis by Snapper, et al (1988, hereinabove cited) indicated that no more than half of the pAL5000 plasmid was necessary to support plasmid replication in BCG. This

T segment presumably carried open reading frames ORF1 and 0RF2, identified By Rauzier, et al.. Gene, Vol. 71, pgs. 315-321

(1988), and also presumably carried a mycobacterial origin of replication. pYTJB12 is then digested with Hpal and EcoRV, a 2586 bp carrying this region or segment pAL5000 is removed and ligated to PvuII digested pYUB8. Plasmid pYUBΘ (a pBR322 derivative) includes an E. coli replicon and a kan (aph) gene. Ligation of the 2586 bp pYUB12 fragment to PvuII digested pYUB8 results in the formation of pYUB53, as depicted in Figure 2. Transformation of pYUB53 confirmed that the EcoRV-Hpal fragment, designated

M.rep, was capable of supporting autonomous replication in BCG.

Plasmid pYUB53 was then digested with AatI, EcoRV, and PstI in order to remove the following restriction sites:

AatI 5707

EcoRI 5783

BamHI 5791

Sail 5797

PstI 5803

PstI 7252

Sail 7258

BamHI 7264

EcoRI 7273

Clal 7298

HindiII 7304; and

EcoRV 7460

Fragment ends are then flushed with T4 DNA polymerase and religated to form plasmid pYUB125, construction of which is shown in Figure 3.

2. Elimination of superfluous vector DNA from PYUB125

792 bases of the tet gene, which had been inactivated by prior manipulations, was eliminated by a complete Narl digest, gel purification of the 6407 bp fragment, and ligation/recirculation, transformation of E. coli strain HB101, and selection of Kan transformants. The construction of resulting plasmid, pMVlOl, is schematically indicated in Figure

4, and the EtøA sequence of pMVlOl, which includes markings of regions which will be deleted, and of mutations, as hereinafter described, is shown in Figure 5.

3. Elimination of undesirable restriction sites in aph

(kan ) gene.

To facilitate future manipulations, the HindiII and Clal restriction sites in the aph gene were mutagenized simultaneously by polymerase chain reaction (PCR) mutagenesis according to the procedure described in Gene, Vol. 77 pgs. 57-59 (1989). The bases changed in the aph gene were at the third position of codons (wobble bases) within each restriction site and the base substitutions made were designed not to change the amino acid sequence of the encoded protein.

Separate PCR reactions of plasmid pMVlOl with primers ClaMut-Kan + HindRMut-Kan and HindFMut-Kan + Bam-Kan were performed at 94^βC (1 min.), 50°C (1 min.), and 72°C (1 min.) for 25 cycles. The PCR primers had the following base sequences:

ClaMut-Kan

CTT GTA TGG GAA CCC CC

HindRMut-Kan

GTG AGA ATG GCA AAA GAT TAT GCA TTT CTT TCC AG

HindFMut-Kan

GTC TGG AAA GAA ATG CAT AAT CTT TTG CCA TTC TCA CCG G

Bam-Kan

CGT AGA GGA TCC ACA GGA CG The resulting PCR products were gel purified and mixed and a single PCR reaction without primers was performed at 94^®C (1 min.), 72°C (1 min.) for 10 cycles. Primers ClaMut-Kan and Bam-Kan were added and PCR was resumed at 94°C (1 min.), 50^βC (1 min.), and 72^βC (2 min.) for 20 cylces. The resulting PCR product (Kan. mut) was digested with BamHI and gel purified. Plasmid pMVlOl was digested with Clal and cohesive ends were filled in by Klenow + dCTP + dGTP. Klenow was heat inactivated

SUBSTITUTE SHEET and the digest was further digested with BamHI. The '5232 base pair fragment was gel purified and mixed with fragment Kan.mut and ligated. The ligation was transformed into E. coli strain p

HB101 and Kan colonies were screened for plasmids resistant to Clal and HindiII digestion. Such plasmids were designated as pMVUO, which is depicted in Figure 4.

4. Elimination of sequences not necessary for plasmid replication in mycobacteria.

Plasmid pMVUO was resected in separate constructions to yield plasmids pMVlll and pMV112. In one construction, pMVUO was digested with Narl and Ball, the ends were filled in, and a 5296 base pair fragment was ligated and recircularized to form pMVlll. In another construct, pMVUO was digested with Ndel and SplI, the ends were filled in, and a 5763 base pair fragment was ligated and recircularized to form pMV112. Schematics of the constructions of pMVlll and pMV112 are shown in Figure 6. These constructions further eliminated superfluous E. coli vector sequences derived from pALSOOO not necessary for mycobacterial replication. Cloning was performed in E. coli. Plasmids pMVlll and pMV112 were tested for the ability to replicate in M. smeqmatis. Because both plasmids replicated in M. smeqmatis the deletions of each plasmid were combined to construct pMV113. (Figure 6).

To construct pMV113, pMVlll was digested with BamHI and EcoRI, and a 1071 bp fragment was isolated. pMV112 was digested with BamHI and EcoRI, and a 3570 bp fragment was isolated, and then ligated to the 1071 bp fragment obtained from pMVlll to form pMV113. These constructions thus defined the region of pAL5000 necessary for autonomous replication in mycobacteria as^'no larger than 1910 base pairs.

5. Mutaqenesis of restriction sites in mycobacterial replicon.

To facilitate further manipulations of the mycobacterial replicon, PCR mutageneais was performed as above to eliminate the Sal I, EcoRI, and Bglll sites located in the open reading frame known as ORFi of pAL5000. PCR mutageneais was performed at wobble bases within each restriction site and the base substitutions were designed not to change the amino acid sequence of the putative encoded ORFI protein. The restriction sites were eliminated one at a time for testing in mycobacteria. It was possible to eliminate the Sail and EcoRI without altering replication in M. smeqmatis. In one construction PCR mutagenesis was performed at EcoRI1071 of pMV113 with primers Eco Mut - M.rep and Bam-M.rep to form pMV117, which lacks the EcoRI1071 site.

Primer Eco Mut - M.rep has the following sequence:

TCC GTG CAA CGA GTG TCC CGG A; and Bam-M.rep has the following sequence:

CAC CCG TCC TGT GGA TCC TCT AC.

In another construction, PCR mutagenesis was performed at the Sail 1389 site with primer Sal Mut - M.rep and Bam-M.rep to form pMV119, which lacks the Sail 1389 site. Primer Sal Mut- M.rep has the following sequence:

TGG CGA CCG CAG TTA CTC AGG CCT. pMV117 was then digested with ApaLI and Bglll, and a 3360 bp fragment was isolated. pMV119 was digested with ApaLI and Bglll, and a 1281 bp fragment was isolated and ligated to the 3360 bp fragment isolated from pMV117 to form pMV123. A schematic of the constructions of plasmids pMV117, pMV119, and pMV123 is shown in Figure 7. Elimination of the Bglll site, however, either by PCR mutagenesis or Klenow fill in, eliminated plasmid replication in mycobacteria, thus suggesting that the Bglll site is in proximity to, or within a sequence necessary for mycobacteria plasmid replication.

6. Construction of pMV200 series vectors.

To facilitate manipulations of all the components necessary for plasmid replication in E. coll and mycobacteria, (E. rep. and p M. rep.) and selection of recombinantβ (Kan ), cassettes of each component were constructed for simplified assembly in future

SUBSTITUTE SHEET vectrs and to include a multiple cloning site (MCS) containing unique restriction sites and transcription and translation terminators. The cassettes were constructed to allow directional cloning and assembly into a plasmid where all transcription is unidirectional. p

Kan Cassette

A DNA cassette containing the aph (Kan ) gene was constructed by PCR with primers Kan 5' and Kan3' . An Spel site was added to the 5' end of the PCR primer Kan3' , resulting in the formation of a PCR primer having the following sequence:

CTC GAC TAG TGA GGT CTG CCT CGT GAA G.

Bam HI + Nhel sites were added to the 5* end of the primer Kan5* , resulting in the formation of a PCR primer having the following sequence:

CAG AGG ATC CTT AGC TAG CCA CT GAC GTC GGG G.

PCR was performed at bases 3375 and 4585 of pMV123, and

BamHI and Nhel sites were added at base 3159, and an Spel site was added at base 4585. Digestion with BamHI and Spel, followed p by purification resulted in a 1228/2443 Kan cassette bounded by

BamHI and Spel cohesive ends with the direction of transcription for the aph gene proceeding from BamHI to Spe I.

E. rep, cassette

A DNA cassette containing the ColEI replicon of pUC19 was constructed by PCR with primers E.rep/Spe and E.rep Mlu. An Spel site was added to the 5* end of PCR primer E.rep/Spe and an Mlul site was added to the 5' end of PCR primer E.rep./Mlu. The resulting primers had the following sequences:

E.rep./Spe

CCA CTA GTT CCA CTG AGC GTC AGA CCC

E.rep. Mlu

GAC AAC GCG TTG CGC TCG GTC GTT CGG CTG.

PCR was performed at bases 713 and 1500 of pUC19, and an Mlul site was added to base 713, and a Spel site was added to base 1500. Digestion with Mlul and Spel, followed by purification resulted in an E.rep. cassette bounded by Spel and

Mlul cohesive ends with the direction of transcription for RNA I and RNA II replication primers proceeding from Spel to Mlul.

M.rep. cassette

A DNA cassette containing sequences necessary for plasmid replication in mycobacteria was constructed by PCR of pMV123 with primers M.rep/Mlu and M.rep/Ba . An Mlul site was added to the 5' end of PCR primer M.rep/Mlu. A BamHI site was added to the 5' end of PCR primer M/rep/Bam. The resulting PCR primers had the following base sequences:

M.rep./Mlu

CCA TAC GCG TGA GCC CAC CAG CTC CG .re ./Bam

CAC CCG TCC TGT GGA TCC TCT AC

PCR was performed at bases 134 and 2082 of pMV123. An Mlul sited was added to base 2082. Digestion with BamHI and Mlul, followed by gel purification resulted in a 1935 base pair DNA cassette bounded by Mlul and BamHI cohesive ends with the direction of transcription for the pAL5000 ORF1 and ORF2 genes proceeding from Mlul to Bam HI. p The Kan , E.rep, and M.rep PCR cassettes were then mixed in equimolar concentrations and ligated, and then transformed in E. p coli strain HB101 for selection of Kan trans ormants. Colonies were screened for the presence of plasmids carrying all three cassettes after digestion with BamHI + Mlul + Spel and designated pMV200. An additional restriction site, Ncol, was eliminated from the M.rep cassette by digestion of pMV200 with Ncol, fill in with Klenow, and ligation and recircularization, resulting in the formation of pMV201. A schematic of the formation of pMV200 from pMV123 and pUC19, and of pMV201 from pMV200, is shown in Figure

8. Plasmids pMV200 and pMV201 were transformed into M. smeqmatis p and BCG. Both plasmids yielded Kan transformants, thus indicating their ability to replicate in mycobacteria.

suBSTm U A synthetic multiple cloning sequence (MCS) (Figure 9) was then designed and synthesized to facilitate versatile molecular cloning and manipulations for foreign gene expressions in mycobacteria, and for integration into the mycobacterial chromosome. The synthetic MCS, shown in Figure 9, contains 16 restriction sites unique to pMV201 and includes a region carrying translation stop codons in each of three reading frames., and a transcription terminator derived from E. coli 5S ribosomal RNA

(Tl).

To insert the MCS cassette, pMV201 was digested with Narl and Nhel, and the resulting fragment was gel purified. The MCS was digested with HinFI and Nhel and, the resulting fragment was gel purified. The two fragments were then ligated to yield pMV204. A schematic of the construction of pMV204 is shown in

Figure 10.

Plasmid pMV204 was then further manipulated to facilitate removal of the M.rep cassette in further constructions. pMV204 was digested with Mlul, and an Mlul - Not I linker was inserted into the Mlul site between the M.rep and the E.rep to generate pMV206. A schematic of the construction of pMV206 from pMV204 is shown in Figure 11, and the DNA sequence of pMV206 is given in

Figure 12.

7. Construction of expression cassette based on BCG HSP60.

Among the most abundant proteins in mycobacteria is the

HSP60 heat shock protein (also known as the 65 kda antigen).

Because abundance of the HSP60 protein in mycobacteria indicates strong HSP60 gene expression, the sequence controlling HSP60 expression was chosen to control expression of heterologous genes encoding antigens or other proteins in BCG.

The published sequence of the BCG HSP60 gene (Thole, et al,

Infect, and I mun. , Vol. 55, pgs. 1466-1475 (June 1987)), and surrounding sequence permitted the construction of a cassette carrying expression control sequences (i.e., promoter and translation initiation sequences) by PCR. The BCG HSP61 cassette (Figure 13) contains 375 bases 5' to the BCG HSP60 start codon, and 15 base? (5 codons) 3' to the start codon. PCR oligonucleotide primers were then synthesized. Primer Xba-HSP60, of the following sequence:

CAG ATC TAG ACG GTG ACC ACA ACG CGC C was synthesized for the 5' end of the cassette, and primer Bam-HSP61, of the following sequence:

CTA GGG ATC CGC AAT TGT CTT GGC CAT TG was synthesized for the 3' end of the cassette. The primers were used to amplify the cassette by PCR from BCG strain Pasteur chromosomal DNA. The addition of the Bam HI site at the 3' end of the cassette adds one codon (Asp) to the first six codons of the HSP60 gene.

Each of pMV206 and the PCR cassette HSP61 was digested with Xbal and BamHI . The PCR cassette was then inserted between the Xbal and BamHI sites of pMV206, then ligated to form plasmid pMV261. The construction of this plasmid is shown schematically in Figure 15. The restriction sites of the multiple cloning site of pMV261 are shown is Figure 14.

The E. coli lac Z gene was used as a reporter, or marker gene to assay the ability of the HSP61 cassette to express heterologous genes in BCG. A BamHI restriction fragment carrying the lac Z gene was cloned into the Bam HI site of Bam HI digested pMV261, resulting in the formation of pMV261/LZ. A schematic of the construction of pMV261/LZ is shown in Figure 16. The formation of pMV261/LZ results in a fusion between the HSP60 and lac Z genes at the sixth codon of the HSP60 gene and the sixth codon of the lac Z gene. pMV261/LZ was then transformed into E. coli. Blue E. coli colonies were selected on x-gal plates for the presence of pMV261/LZ, thus indicating that the HSP60 promoter and translation initiation sequences were also active in E. coli. pMV261/LZ was then transformed into BCG and plated on Dubos Oleic Agar plates containing x-gal. All BCG colonies resulting

SUBSTITUTE SHE from this transformation exhibited blue color, thus indicating that the lac^" Z gene product (B-galactosidase) was expressed in

BCG. SDS polyacrylamide gel electrophoresis was performed on lysateβ of the pMV261/LZ BCG recobinants, revealing that

B-galactosidase protein was expressed to levels in excess of 10% of total BCG protein (as determined by staining with Coomassie brilliant blue). These data indicated that BCG HSP61 expression cassette was functional in expression vector pMV261, and that substantial expression of a heterologous gene could be achieved using HSP60 - controlled expression in BCG.

Plasmid pMV261/LZ was then shown to replicate autonomously, and express the E. coli B-galactosidase, or lacZ gene, driven by the BCG promoter HSP60, in M. smeqmatis and BCG.

Example 2

Construction of expression cassette based on BCG HSP70.

A partial sequence of the 5' region of the BCG HSF70 gene (which encodes for the BCG HSP70 heat shock protein, also known as the 70 kda antigen) obtained by Dr. Rick Young (MIT) permitted the construction of cassettes carrying expression control sequences (i.e., promoters and translation initiation sequences) by PCR, according to the procedures hereinabove cited. The BCG-HSP71 cassette (Figure 13) contains 150 bases 5' to the BCG-HSP70 start codon and 15 bases (5 codons) 3' to the start codon. Primer Xba-HSP70 was synthesized for the 5' end of the cassette, and primer Bam-HSP71 was synthesized for the 3' end of the cassette. The primers had the following base sequences:

Xba-HSP70 σσc ere TAG ACC CGC ACG ACC AGC GTT AGC

Bam-HSP71

GCT AGG ATC CCC GAC CGC ACG AGC CAT GGT The primers were used to amplify the cassette from BCG subβtrain Pasteur chromosomal DNA. The addition of the Bam HI site at the 3* end of the cassette adds 1 codon (Asp) to the 3' end of the HSP71 expression cassette. Each of the pMV206 and the PCR cassette HSP71 was digested with Xbal and BamHI. The PCR cassette was inserted between the

Xbal and BamHI sites of pMV206, and then ligated to form plasmid pMV271. A schematic of the construction of plasmid pMV271 is shown in Figure 15.

Example 3

Cloning and Expression of Tetanus Toxin Fragment C in BCG.

A gene encoding Fragment C of tetanus toxin was amplified from Clostridium tetani chromosomal DNA by polymerase chain reaction (PCR) . The oligonucleotide primers used for this PCR procedure were synthesized with a Bam HI site (primer CS73) and a Cla I site (primer CS72), such that the resulting tox C PCR fragment carried a Bam HI site at the 5' end and a Cla I site at the 3' end of the tox C.

Primer CS73 has the following sequence: tox C:5' CGG GAT CCT TCA ACA CCA ATT CCA TTT TCT TAT TC Bam HI

Primer CS72 has the following sequence: tox C:3' GCAT CGA TTC ATG AAC ATA TCA ATC TGT TTA ATC Cla I

The tox C PCR fragment was then digested with restriction enzymes Bam HI and Cla I, purified by agarose gel electrophoresis, inserted by ligation between the Bam HI and Cla I sites of extrachromosomal plasmid vectors pMV261 and pMV271 and transformed into E.coli. Recombinant plasmids carrying the tox C gene segment, designated pMV261::tox C (Figure 16) and pMV271::tox C (Figure 17) were transformed into BCG. BCG transofrmants were then analyzed by western blot analysis with polyclonal rabbit sera for expression of the toxC polypeptide (Figure 18), as compared to Fragment C polypeptide purified from C.tetani in amounts of 0.2 ng, 2ng, lOng, and 50ng. Polypeptides that reacted with the anti-tetanus toxin were observed at primary molecular weights of approximately 53kDa, consistent with the size of Fragment C plus 6 codons of the hsp 60 (in pMV261) or hs^p

SUB I E 70 (in pMV271). Additional lower molecular weight immunoreactive polypeptides thought to be proteolytic products were also observed. Immunoreactive polypeptides were not observed in nonrecombinant BCG.

Example 4 BCG transformed with pMV261: tox C was used to immunize NIH/Swiss mice to assess its its efficiency against tetanus toxin. NIH/Swiss mice (5 per group) were immunized with 10 colony-forming units (CFU's) of recombinant BCG vaccine via intradermal (ID), intrapertioneal (IP), or intravenous (IV) injection. A control group of unimmunized mice was also formed. Thirteen weeks after immunization, all mice were challenged by subcutaneous injection in the inguinal fold of 100 LD_ς- units of active tetanus toxin. The mice were monitored for survival several times daily for four days. All mice in the control group died within 24 hours of challenge. The mice immunized with the transformed BCG intravenously had a survival rate of about 20% after 30 hours. All mice which were immunized intravenously died within 40 hours of challenge. The mice which were immunized intradermally had a survival rate of about 75% at 50 hours post-challenge, of about 50% from 60 to 95 hours post-challenge, and of about 25% at 110 hours post-challenge. The mice which were immunized intraperitoneally had a survival rate of about 80% at 50 hours post-challenge and of about 60% from 60 to 110 hours post-challenge. The drop in the survival rates observed in the immunized mice suggests that the NIH/Swiss mouse strain is a "low responder" strain if immunized with BCG. The immunized groups, however, each showed varying degrees of protection from, toxin challenge, while all mice in the control group died within 24 hours of challenge.

It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the^" accompanying claims.

Claims

WHAT IS CLAIMED IS:

1. A method of inducing an immune response to a tetanus toxin in an animal, comprising: administering to an animal mycobacteria transformed with at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin, or a fragment(s) or derivative(s) thereof, said mycobacteria being administered in an amount effective to induce an immune response to a tetanus toxin in said animal.

2. The method of Claim 1 wherein said at least one DNA sequence encodes Fragment C of tetanus toxin, or a derivative or fragment thereof.

3. The method of Claim 1 wherein said mycobacteria are of the species M.bovis-BCG.

4. A composition for inducing an immune response to a tetanus toxin in an animal, comprising: mycobacteria transformed with at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against tetanus toxin, or fragment(s) or derivative(s) thereof; and an acceptable pharmaceutical carrier, said mycobacteria being present in an amount effective to induce an immune response against tetanus toxin in an animal.

5. The composition of Claim 4 wherein said at least one DNA sequence encode* ^"Fragment C of tetanus toxin or a derivative or fragment thereof.

6. The composition of Claim 4 wherein said mycobacteria are of the species M.bovis - BCG.