WO1994014318A1

WO1994014318A1 - Method of protection against streptococcus pneumoniae with transformed mycobacteria

Info

Publication number: WO1994014318A1
Application number: PCT/US1993/012504
Authority: WO
Inventors: David Briles; Charles K. Stover
Original assignee: Medimmune, Inc.; Uab Research Foundation
Priority date: 1992-12-24
Filing date: 1993-12-20
Publication date: 1994-07-07
Also published as: AU5958594A

Abstract

A method of protecting an animal against Streptococcus pneumoniae by administering to the animal mycobacteria which are transformed with DNA which includes at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against Streptococcus pneumoniae. The at least one DNA sequence may encode a surface protein of Streptococcus pneumoniae, and in particular PspA protein or a fragment or derivative thereof. Such mycobacteria may be administered in combination with a pharmaceutical carrier as a vaccine against Streptococcus pneumoniae.

Description

O 94/1431

METHOD OF PROTECTION AGAINST STREPTOCOCCUS PNEUMONIAE WITH TRANSFORMED MYCOBACTERIA

This invention relates to protecting animals against Streptococcus pneumoniae. More particularly, this invention relates to protecting an animal against Streptococcus pneumoniae by administering to an animal mycobacteria transformed with DNA encoding Streptococcus pneumoniae proteins.

In accordance with an aspect of the present invention, there is provided a method of protecting an animal against Streptococcus pneumoniae by administering to an animal mycobacteria transformed with DNA which includes at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against Streptococcus pneumoniae. The mycobacteria are administered in an amount effective to protect an animal against Streptococcus pneumoniae In one embodiment, the at least one DNA sequence encodes a surface protein of Streptococcus pneumoniae or a fragment or derivative thereof. Surface proteins of Streptococcus pneumoniae which may be encoded by the at least one DNA sequence include, but are not limited to, Pneumococcal Surface Protein A (hereinafter sometimes referred to PspA) or a fragment(s) or derivative( s ) thereof

Mycobacteria which may be transformed with the DNA encoding a protein or polypeptide which elicits antibodies against Streptococcus pneumoniae include, but are not limited to, Mycobacterium bovis - BCG, M. smecrmatis, M. avium, M.phlei , M. - -

fortuitu , M. ufu, M.paratuberculos s, M.habana, M. scrofalaceum, M. intracellulare. and M.vaccae. In one embodiment, the mycobacterium is M.bovis - BCG.

In a preferred embodiment, the at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against Streptococcus pneumoniae is contained in a mycobacterial expression vector.

In one embodiment, the mycobacterial expression vector may further include a DNA sequence encoding at least a secretion signal of a lipoprotein, and wherein the mycobacterium expresses a fusion protein of at least the secretion signal of the lipoprotein and the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae. Although certain proteins or polypeptideε which elicit antibodies against a surface protein of Streptococcus pneumoniae, such as, for example, the PspA antigen or a fragment or derivative thereof, are not lipoproteinε, such an expression vector enables the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae to be expressed on the surface of the mycobacterium, whereby the protein or polypeptide becomes more accessible .

It is believed that the signal sequence of the lipoprotein enables the expressed recombinant fusion protein to be modified such that the protein iε expressed at the surface of the bacterium. For example, the fusion protein may include active site(s) for signal peptidase II in the signal sequence portion, which enables lipid acylation of the fusion protein. Such lipid acylation of the fusion protein may enhance the immunogenicity of the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae .

In one embodiment, the DNA sequence encodes at least a secretion signal of a mycobacterial lipoprotein. The mycobacterial lipoprotein may, in one embodiment, be an M. tuberculosis lipoprotein. The M. tuberculosis lipoprotein may be selected from the group consisting of the M.tuberculosis 19 kda antigen and the M. tuberculosis 38 kda antigen.

Other lipoproteins, of which at least the secretion signal may be encoded by the DNA sequence include, but are not limited to, Braun's lipoprotein of E. coli, S. marcescens, E. amylosora, M. morganii, and P. mirabilis, the TraT protein of E. coli and Salmonella; the penicillinase (PenP) protein of B. licheniformis and B. cereus and S. aureus; pullulanase proteins of Klebsiella pneumoniae and Klebsiella aeroqenese; E. coli lipoproteins lpp-28, Pal, RplA, RplB, Os B, NlpB, and 0rll7; chitobiase protein of V. harseyi ; the β-1, 4-endoglucanase protein of Pseudo onas solanacearum, the Pal and Pep proteins of H. influenzae; the OprI protein of P. aeruginosa; the MalX and AmiA proteins of S. pneumoniae; the 34 kda antigen and TpmA protein of Treponema pallidum; the P37 protein of Mycoplasma hyorhinis; and the 17 kda antigen of Rickettsia rickettsii. It is to be understood, however, that the scope of the present invention iε not to be limited to secretion signalε of any particular lipoprotein or lipoproteins.

In one embodiment, the DNA sequence may further include DNA which encodes all or a portion of the lipoprotein. Thus, in such an embodiment, the fusion protein which is expressed by the bacterium is a fusion protein of the secretion signal of the lipoprotein, all or a portion of the lipoprotein, and the protein or polypeptide or peptide which elicits antibodies against Streptococcus pneumoniae .

The DNA sequence, which encodes at least the secretion signal of the lipoprotein, and the DNA which encodes the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae, are under the control of a suitable promoter. In one embodiment, the promoter may be the 19 kda antigen promoter or the 38 kda antigen promoter of M. tuberculosis if DNA encoding the secretion signal of one of these antigens is employed. Alternatively, the promoter may be a mycobacterial promoter other than the 19 kda and 38 kda M.tuberculosis antigen promoters, or a mycobacteriophage promoter.

Mycobacterial and mycobacteriophage promoters which may be employed include, but are not limited to, mycobacterial promoters such as the BCG HSP60 and HSP70 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, D29, and TM4 promoters. In one embodiment, the promoter is a mycobacterial heat shock protein promoter such as HSP60 or HSP70. It iε also contemplated that, within the scope of the present invention, that the expression vector include mycobacterial and mycobacteriophage promoters such as those hereinabove described, without including the DNA encoding at least the secretion signal of a lipoprotein.

Example of expression vectors including the mycobacterial promoters and mycobacteriorphage promoters hereinabove described are further described in application Serial No. 642,017, filed January 16, 1991, which iε 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 a preferred embodiment, the transcription initiation site, the riboso al 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 Streptococcus pneumoniae, and the transcription termination site, may be of mycobacterial origin, or of other bacterial origin, or may be synthetic in nature, or such stop codon and transcription termination site may be those of the DNA encoding the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae.

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 Streptomyces origin of replication, or a streptococcal 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 sequence encoding the protein or polypeptide which elicitε antibodies against Streptococcus pneumoniae is inserted in the multiple cloning site.

In another embodiment, 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 DNA sequence encoding at least the secretion signal of a lipoprotein and a DNA sequence encoding a protein or polypeptide or peptide which elicits antibodies against Streptococcus pneumoniae stably into mycobacteria, in which the DNA sequences may be expressed. When a shuttle phasmid, which replicates aε a phasmid in bacteria and a phaσe in mycobacteria, is employed, integration of the phasmid. which includes the DNA sequence encoding at least the secretion signal of a lipoprotein, and the DNA sequence encoding a protein or polypeptide or peptide which elicits antibodies against Streptococcus pneumoniae, into the mycobacterial chromosome, occurs through site-specific integration. The DNA seqeunces are replicated as part of the chromosomal DNA. When a bacterial-mycobacterial shuttle plasmid - -

is employed, the DNA sequences are stably maintained extrachormosomally in a plasmid. Expression of the DNA sequences occur extrachromosomally (e.g., episomally) . For example, the DNA sequences are cloned into a shuttle plasmid and the plasmid is introduced into a mycobacterium such as those hereinabove described, wherein the plasmid replicates episomally. 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 addition to the DNA sequence encoding at least the secretion signal of a lipoprotein and the DNA sequence encoding a protein or polypeptide which elicits antibodies against Streptococcus pneumoniae, and the mycobacterial promoter for controlling expression of the DNA encoding the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae, 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, D29, and TM4 mycobacteriophages; the lambda phage of E. coli; the toxin phages of Corynebacteria; phageε of Actinomycetes and Norcardia; the fC31 phage of Streptomyces; 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.

The DNA encoding the attP site and the integrase provides for an integration event which iε 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 DNA which is a phage DNA portion encoding bacteriophage integration into a mycobacterium chromosome are further described in Application Serial No. 869,330, filed April 15, 1992, which is a continuation-m-part of Application Serial No. 553,907, filed July 16, 1990, now abandoned. The contents of Application Serial No. 869,330 are incorporated by reference.

The mycobacterial expression vectors include DNA which encodes a protein or polypeptide, which, as hereinabove stated, elicits antibodies against Streptococcus pneumoniae. Thus, the transformed mycobacteria may be employed as part of a pharmaceutical composition, such aε a vaccine against Streptococcus pneumoniae, which includes the transformed mycobacteria, and an acceptable pharmaceutical carrier. Acceptable pharmaceutical carriers include, but are not limited to, 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 m 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 m the form of an injectable dose and may be administered intramuscularly, intravenously, orally, mtradermally, or by subcutaneous administration

The mycobacteria are administered in an effective amount. In general, the mycobacteria are administered in an amount of from about 1 x 10 to about 1 x 10 colony forming units (CFU's) per dose. O 94/1431 - -

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.

In another embodiment, the mycobacterial expression vector may contain DNA which encodes all or a portion of a mycobacterial excretion protein, as well as the DNA which encodes a protein or polypeptide which elicits antibodies against Streptococcus pneumoniae. The mycobacterium expresses a fusion protein of the mycobacterial excretion protein or a portion thereof, and the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae. Such an expression vector enables the protein or polypeptide to be excreted from the mycobacterium. Examples of mycobacterial excretion proteins which may be encoded, include, but are not limited to, the α-antigen of M. tuberculosis and BCG.

The invention will now be described with respect to the following examples; however, the scope of the present invention is not intended 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. 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 segment presumably carried open reading frames 0RF1 and ORF2, identified by Rauzier, et al., Gene, Vol. 71, pgs. 315-321 (1988), and also presumably carried a mycobacterial origin of replication. pYUB12 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 , aε depicted in Figure 2. Transformation of pYUB53 confirmed that the EcoRV-Hpal fragment, designated M.rep, was capable of supporting autonomouε replication in BCG.

Plasmid pYUB53 waε 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 729Θ

Hindi11 7304; and

EcoRV 7460 9 /

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 DNA 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 Hindlll 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 HmdFMut-Kan + Barn-Kan were performed at 94°C (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 GCC CC HindRMut-Kan

GTG AGA ATG GCA AAA GAT TAT GCA TTT CTT TCC AG HmdFMut-Kan

GTC TGG AAA GAA ATG CAT AAT CTT TTG CCA TTC TCA CCG G Barn-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 mm. ) for 10 cycles. Primers ClaMut-Kan and Barn-Kan were added and PCR was resumed at 94°C (1 mm. ), 50°C (1 mm. ), and 72°C (2 mm. ) 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 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 HBlOl and Kan colonies were screened for plasmids resistant to Clal and H dlll digestion Such plasmids were designated aε pMVllO, which is depicted in Figure 4

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

Plasmid pMVllO was resected in separate constructions to yield plasmids pMVlll and pMV112. In one construction, pMVllO was digested with Narl and Ball, the ends were filled in, and a 5296 base pair fragment was ligated and recirculaπzed to form pMVlll In another construct, pMVllO was digested with Ndel and SplI, the endε 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 pAL5000 not necessary for mycobacterial replication. Cloning was performed in E. coli . Plasmids pMVlll and pMV112 were tested for the ability to replicate m 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 9 /1 - -

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 paris.

5. Mutaqenesis of restriction εites in mycobacterial replicon.

To facilitate further manipulations of the mycobacterial replicon, PCR mutagenesis was performed as above to eliminate the Sal I, EcoRI, and Bglll sites located in the open reading frame known as ORF1 of pAL5000. PCR mutagenesis 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 ORF1 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.

PNV117 was then digested with Apal 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. coli and mycobacteria, (E. rep. and

M. rep. ) and selection of recombmants (Kan ), cassettes of each component were constructed for simplified assembly in future vectrε and to include a multiple cloning site (MCS) containing unique restriction sites and transcription and translation terminators. The casεetteε were constructed to allow directional cloning and aεsembly into a plasmid where all transcription is unidirectional . p Kan Cassette p

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 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 DΝA cassette containing sequences necessary for plasmid replication in mycobacteria was conεtructed 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

M. rep. /Bam

CAC CCG TCC TGT GGA TCC TCT AC

PCR was performed at baseε 134 and 2082 of pMV123. An Mlul εite was added to base 2082. Digestion with BamHI and Mlul, followed by gel purification resulted in a 1935 base pair DΝA cassette bounded by Mlul and BamHI cohesive ends with the direction of transcription for the pAL5000 ORF1 and ORF2 genes proceeding from Mlul to BamHI .

The Kan , E.rep, and M.rep PCR cassettes were then mixed in equimolar concentrations and ligated, and then transformed in E. coli strain HBlOl 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. Plasmidε pMV200 and pMV201 were transformed into M. smegmatis and BCG. Both plasmids yielded Kan transformants, thus indicating their ability to replicate in mycobacteria.

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 reεtriction sites unique to pMV201 and includes a region carrying translation stop codons in each of three reading frames, and a Tl transcription terminator derived from E. coli rrnAB ribosomal RNA operon.

To insert the MCS casεette, pMV201 was digested with Narl and Nhel, and the resulting fragment was gel purified. The MCS was digeεted with HinPI and Nhel and, the resulting fragment waε 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 waε digeεted 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. Insertion of BCG HSP60 promoter sequence.

The published sequence of the BCG HSP60 gene (Thole, et al. , Infect, and Immun. , Vol. 55, pgs. 1466-1475 (June 1987)), and surrounding sequence permitted the construction of an HSP60 - -

promoter fragment by PCR. The 251 bp HSP60 promoter fragment (Figure 13, and as published by Stover, et al . (1991)) was amplified by PCR with primers including added Xbal and Nhel sites. The PCR HSP60 fragment is then digested with Xbal and Nhel, and is ligated into Xbal digested pMV206 to form pRB26 (Figure 14) .

8. Insertion of DNA encoding the 19 kda M. tuberculosis signal sequence into mycobacterial expression vector.

The sequence of the 19 kda M. tuberculosis gene is given in Ashbridge, et al . , Nucleic Acids Research, Vol. 17, pg. 1249 (1989). The 19 kda antigen gene ribosomal binding site, start codon, and signal sequence from M. tuberculosis chromosomal DNA were amplified by PCR with nucleotide primers. The resulting 153 bp fragment (Figure 15) obtained by PCR includes added Bglll (5' ) and BamHI: EcoRI sites (3' ). This fragment contains the entire 5' region of the 19 kda gene up to the 27th codon with the exception of the promoter sequence. The PCR fragment is digested with Bglll and EcoRI and ligated into BamHI-EcoRI digested pRB26 to form p2619S (Figure 16).

9 • Insertion of DNA Encoding PspA Fragment

The PspA antigen of S. pneumoniae is described in Briles, et al . , Reviewε of Infectiouε Diseases, Vol. 10, Supplement 2, pgs. 5372-5374 (July-August 1988), and in Crain, et al. , Infect, and Im un. , Vol. 58, No. 10, pgs. 3293-3299 (October 1990). The antigen has been shown to elicit protective immune responseε to challenge with virulent S. pneumoniae in mice. The PspA antigen has been divided into four structural domains: (i) the N-terminal 18 amino acid signal peptide; (ii) the N-terminal -helical 33kda protective region; (iii) a central proline-rich region; and (iv) the C-terminal repetitive region. (McDaniel, et al . , Infect, and Immun. , Vol. 59 pgs. 222-228 (Jan. 1991)).

The 5' half of the PspA gene (which encodes the pspA antigen of S. pneumoniae) , without the region encoding the N-terminal 18 amino acid signal peptide (known as PspA3), was amplified from S__^ pneumoniae chromosomal DNA by PCR. The resulting fragment, obtained by PCR, is an 896 bp fragment and includes added BamHI and Sail sites. The DNA sequence of this fragment is shown in Figure 17. Thiε PCR fragment encodeε the PspA protective region. The PCR fragment was then digested with BamHI and Sail and cloned into BamHI and Sail digested p2619S to form p2619S: :PspA33A. (Figure 18. )

Example 2

PNV206 was conεtructed aε hereinabove described in Example 1.

The published sequence of the BCG HΞP60 gene (Thole, et al, Infect, and Immun. , Vol. 55, pgs. 1466-1475 (June 1987)), and surrounding sequence permitted the construction of a cassette carrying expression control sequences (i.e., promoter, ribosomal binding site, and translation initiation sequenceε as published in Stover, et al . (1991)) by PCR. The BCG HSP61 cassette (Figure 19) contains 375 bases 5" to the BCG HSP60 start codon, and 15 baseε (5 codonε) 3' to the start codon. PCR oligonucleotide primers were then syntheεized 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 TCT CTT GGC CAT TG waε εyntheεized for the 3' end of the caεsette. 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 codσn (Asp) to the first six codons of the HSP60 gene.

Each of pMV206 and the FCR 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 - -

PNV261 . The construction of thiε plaεmid is shown schematically in Figure 20.

The PCR PspA protective region sequence was constructed aε hereinabove described in Example 1, digested with BamHI and Sail and was cloned into BamHI and Sail digested pMV261 to form pMV261: :PspA33A. (Figure 21.)

Example 3 pRB26 was constructed as hereinabove described in Example 1. The 5' half of the PspA gene was obtained by PCR as described in Example 1, except that the resulting fragment also included DNA encoding the N-terminal 18 am o acid signal peptide (known as PspA3 ) and the riboεomal binding εite of PspA at the 5' end, addition to the 896 bp fragment described in Example 1. The resulting fragment includes added BamHI and Sail sites. This PCR fragment was then digested with BamHI and Sail and cloned into BamHI and Sail digested pRB26 to form pRB26: :PspA33B.

Example 4 pMV206 waε constructed as hereinabove described in Example 1.

A partial sequence of the 5' region of the BCG HSP70 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 expresεion control sequences (i.e., promoters and translation initiation sequences) by PCR, according to the procedures hereinabove cited. The BCG-HSP71 cassette (Figure 22) 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 waε synthesized for the 3' end of the cassette. The primers had the following base sequences-

Xba-HSP70

GGC CTC 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 substrain 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 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 iε shown in Figure 23.

The PCR PspA 896 bp protective region sequence was conεtructed as hereinabove described in Example 1, digested with BamHI and Sail, and waε cloned into Bam HI and Sail digested pMV271 to form pMV271 : :PspA33A.

Example 5 pMV206 was constructed as described in Example 1.

The 19 kda M. tuberculosis antigen gene promoter, ribosomal binding site, start codon, and secretion signal was amplified by PCR with nucleotide primers. The PCR fragment includes added Xbal and BamHI sites. This sequence, shown in Figure 24, which is 286 bp in length, includes the entire published 5' region of the 19 kda gene up to the 27th codon. The PCR fragment waε then digested with Xbal and BamHI, and ligated into Xbal and BamHI digested pMV206 to form pl9PS (Figure 25). The PCR PspA 896 bp protective region sequence of Example 1 was digeεted with BamHI and Sail, and was cloned into BamHI and Sail digested pl9PS to form pl9PS: :PspA33A. (Figure 26. )

Example 6 The gene sequence for the M. tuberculosis 38 kda antigen is given in Andersen, et al., Infection and Immunity, Vol. 57, No. 8, pgs. 2481-2488 (Aug. 1989). A DNA sequence encoding the 38 - -

kda antigen promoter, ribosomal binding site, start codon, and secretion signal, obtained from M. tuberculosis chromosomal DNA, and containing the entire 5' sequence up to the 45th codon, was amplified by PCR with nucleotide primers. The resulting PCR fragment includes added Xbal and BamHI sites. The PCR fragment, 297 bp in length, and shown in Figure 27, was digested with Xbal and BamHI, and ligated into Xbal and BamHI digested pMV206 to form p38PS (Figure 28). The PCR PspA 896 bp protective region sequence is digested with BamHI and Sail, and was cloned into BamHI and Sail digested p38PS to form p38PS: :PspA33A. (Figure 29) .

Example 7

BCG Pasteur organisms were transformed by electroporation with one of the following vectors: pMV261: :lacZ (Control). pMV261: :pspA33A pMV271: :pspA33A pRB26: :pspA33B p2619S: :pspA33A pl9PS: :pspA33A; and p38PΞ: :pspA33A.

Kanamycin resistant transformants were selected on plates containing 20 μg/ml kanamycin, and then grown in liquid media to late log phase. Recombinant BCG lyεates were then prepared, and subjected to SDS-polyacrylamide gel electrophoresis (Figure 30a), and subsequent Western blot analysis with monoclonal antibodies specific for the PspA protein' (Figure 30b). As shown in Figures 30a and 30b, recombinant BCG expressed PspA segments migrated at apparent molecular weights consistent with previous reportε, and that of the purified PspA33 protein (shown in amounts of lOOng and 20ng) .

Example 8 O 94/14318 - -

BCG Pasteur organisms were transformed with one of the following vectors: pMV261: :OspA

PNV271 : :PspA33A pRB26: :PspA33B pl9PS: :PspA33A; or p38PS: :PspA33A. pMV261: :OspA iε a vector which expresseε the OspA antigen of Borrelia burgdorferi , the causative agent of Ly e disease. pMV261: :OspA is further described in co-pending United States patent application Serial No. 780,261, filed October 21, 1991.

Strain 129/J mice were divided into groups of five, and each of the mice in each group were immunized with 10 CFU'ε of recombinant BCG organiεmε transformed with one of the vectors hereinabove described. Pooled sera taken at 8 and 12 weeks were diluted 200 fold, and were analyzed by ELISA with purified PspA recombinant antigen (33 kda variable protective region.) As shown Figure 31, only mice immunized with recombinant BCG expressing the PspA gene mounted an immune response against PspA.

Example 9

BCG organisms were transformed with p2619S::PspA or pMV261 : :PspA. BCG transformed with p2619S: :PspA were subjected to analysis with the Triton X-114 phase partitioning syεtem and Western blotting.

The analysis waε carried out aε follows: The transformed BCG cellε were cultured, and the cells were then sedimented from the cultures. The cells were then εuεpended in phosphate buffered saline (PBS), and cell suspensions were normalized to equivalent densities. The cellε were disrupted by sonication, the cell envelopes were sedimented, and the supernatant (a Cytoεol-enπched fraction) was saved. The cell envelopes were resuspended in PBS, and membranes were solubilized at 4°C by the addition of Triton X-114 to 2% (vol./vol. ) . Inεoluble material - -

(a cell wall-enriched fraction) was sedimented, and the supernatant (membrane-enriched fraction) was removed. Triton X-114 was added to the Cytosol-enriched fraction. After brief warming of the Triton X-114 solutions at 37°C, separation of aqueous and detergent phases was achieved by a short centrifugation These two phases were back-extracted three timeε, and proteins in representative samples were precipitated by the addition of acetone A portion of each culture supernatant was concentrated by an ultrafiltration device (Centrιcon-30, Amicon). Samples representing culture volume equivalents were processed by SDS-PAGE, transferred to nitrocellulose, and Western blotted with anti-PspA antibody. Filter-bound antibody was visualized with an enhanced chemiluminescence. system (Amersham).

Such analysiε εhowed that there waε high level expression (greater than 1% of total cell protein) of the PspA protective region The analysis also showed that the PspA protein was localized in the membrane fractions, thus indicating that fusion of the PspA gene to a lipoprotein secretion signal could result in the expression of a normally nσn-lipoprotein (PspA) as a recombinant lipoprotein that is associated with the bacterial membrane

BCG transformed with p2619S- :PspA and pMV261: :PspA were subjected to fluorescent antibody cell sorting (FACS). Fluorescent antibody labelling of recombinant BCG cell surfaceε with monoclonal antibodieε εpecific for PspA indicated that PspA is expressed at the surface of BCG only when PspA is fused to a lipoprotein signal sequence (in this case for the 19 kda M tuberculosis antigen) PspA is expressed on the surface by p2619S- :PspA, but not by pMV261 -PεpA

Example 10

Mice of the strains 129J, BALB/C, C3H/He, or C57 were immunized with 10 colony forming units of BCG organisms transformed with pMV261 -OspA (control), pMV261 : :pspA33A; pMV271: :pspA33A, pRB26: :pspA33B, pl9PS: :pspA33A, or p2619S: :pspA33A. The mice were given booster immunizations of the same dose 17 weeks after the initial immunization. Anti-PspA titers were measured by ELISA with purified PspA at 16 weeks after the initial immunization and at two weeks after the booster immunization (19 weeks after the initial immunization). Antibody end point titers are given in Table I below.

Table_I

Vector Mouse Strain Titer

PNV261 :OspA 129J pMV261 :OspA BALB/C pMV261 :OεpA C3H/He pMV261 :OspA C57 pMV261 :pεpA33A 129J pMV261 :pspA33A BALB/C pMV261 :pεpA33A C3H/He pMV261 :pspA33A C57 pMV271 :pspA33A 129J

PNV271 :pεpA33A BALB/C pMV271 :pspA33A C3H/He pMV271 :pspA33A. C57 pRB26 pεpA33B 129J pRB26 pspA33B BALB/C pRB26 pspA33B C3H/He pRB26 pspA33B C57 pl9PS pspA33A I29J pl9PS pεpA33A BALB/C pl9PS pεpA33A C3H/He pl9PS pεpA33A C57 p2619S :pspA33A 129J p2619S :pspA33A BALB/C p2619S :pspA33A C3H/He p2619S :p.spA33A C57

At three weeks after the booster immunization (20 weeks after the initial immunization), the mice immunized with BCG transformed with pMV261 : :ospA, pMV261 : :pspA33A, or p2619S: :pspA33A were exsanguinated and the sera were diluted to 1:3, 1:12, and 1:48. In order to test for protective immune responseε, two XID mice for each group of immunized and exsanguinated mice were given lOOμl of diluted sera intraperitoneally, and then were challenged within one hour with 2,000 colony forming units of S.pneumoniae - WU2 strain IV. The mice were observed for 20 days. Protection from death due to the pneumococcal challenge was defined as survival to 20 days following the challenge. The greateεt antisera dilution at which protection waε observed is given in Table II below.

Table II

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 protecting an animal against Streptococcus pneumoniae, comprising: administering to an animal mycobacteria transformed with DNA which includes at least one DNA sequence which encodes a protein or polypeptide which elicitε antibodieε againεt Streptococcuε pneumoniae, εaid mycobacteria being administered in an amount effective to protect an animal againεt Streptococcus pneumoniae .

2. The method of Claim 1 wherein said at least one DNA sequence encodes a surface protein of Streptococcus pneumoniae or a fragment or derivative thereof.

3. The method of Claim 2 wherein said surface protein is Pneumococcal Surface Protein A or a fragment(ε), or derivati e( s ) thereof.

4. The method of Claim 1 wherein εaid DNA which encodes a protein or polypeptide which elicitε antibodies against Streptococcuε pneumoniae iε contained in a mycobacterial expression vector.

5. The method of Claim 4 wherem said mycobacterial expresεion vector further includes a DNA sequence encoding at least a secretion signal of a lipoprotein, whereby said mycobacteria express a fusion protein of at least the secretion signal of a lipoprotein and the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae.

6. The method of Claim 4 wherein said mycobacterial expression vector further includes a promoter selected from the group consisting of mycobacterial promoters and mycobacteriophage promoters

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

8. A composition for protecting an animal against Streptococcus pneumoniae, comprising: - -

ycobacteria transformed with DNA which includes at least one DNA sequence which encodes a protein or polypeptide which elicits antibodies against Streptococcus pneumoniae; and an acceptable pharmaceutical carrier, said mycobacteria being present in an amount effective to protect an animal against Streptococcus pneumoniae.

9. The composition of Claim 8 wherein εaid at least one DNA sequence encodes a surface protein of Streptococcus pneumoniae or a fragment or derivative thereof

10. The composition of Claim 9 wherem said surface protein lε Pneumococcal Surface Protein A or a fragment(ε) or derivative( ε) thereof.

11. The composition of Claim 8 wherem εaid DNA which encodeε a protein or polypeptide which elicits antibodieε againεt Streptococcus pneumoniae iε contained in a mycobacterial expression vector.

12. The compoεition of Claim 11 wherein εaid mycobacterial expression vector further includes a DNA sequence encoding at least a secretion signal of a lipoprotein, whereby said mycobacteria expresε a fusion protein of at least the secretion signal of a lipoprotein and the protein or polypeptide which elicits antibodies against Streptococcus pneumoniae.

13. The composition of Claim 11 wherem said mycobacterial expression vector further includes a promoter selected from the group consisting of mycobacterial promoterε and mycobacteriophage promoterε.

14. The composition of Claim 8 wherein said mycobacteria are of the species M.bovis-BCG.