Patent Abstract:
recombinant immunogenic compositions , and methods for the manufacture and use , are provided for the prevention and treatment of intracellular pathogen diseases in humans and animals . the recombinant immunogenic compositions express high levels of recombinant proteins in vectors that do not harbor an antibiotic resistance marker .

Detailed Description:
unmarked strains of live vaccine vectors have been produced previously by various means . the most common method has relied on expression of the desired antigen from a plasmid using a balanced - lethal plasmid stabilization system that allows antibiotic resistance markers to be eliminated from the plasmid . plasmid expression systems are often used to obtain high expression levels , as expression of genes integrated into the chromosome is frequently low level . however , genes integrated into the chromosome of live vaccine vectors are regarded as more stable than plasmid based genes . in contrast to the technology cited above , the present invention provides methodology for obtaining an unmarked strain and allows high expression from a gene integrated into the chromosome . thus , the present disclosure provides both the stability advantage of chromosomal integration and the advantage of high expression of a recombinant antigen . newer , site - specific integration methods for incorporating genes into the chromosome without antibiotic resistance genes have been developed , but these methods have been developed using escherichia coli and may not work in unrelated bacteria without a great deal of engineering . furthermore , these methods are limited to a single site of integration on the chromosome . although in the present disclosure the integration is located at a single site ( in the glna1 locus ), the method could be used to integrate genes in many locations on the chromosome , wherever an essential gene can be deleted and subsequently restored . multiple integrations could also be done sequentially with this method , incorporating multiple genes at different locations on the chromosome . in previous studies , genes have been incorporated into the his locus of salmonella via homologous recombination ( without antibiotic resistance genes ) for the purpose of expression in a live vaccine vector . in one study , expression from the chromosome was shown to be far more stable than expression from a plasmid , but expression was also greatly reduced , thus weakening the immune response to the vaccine . in contrast , the present disclosure combines the stability of chromosomal integration with high expression of a recombinant antigen . furthermore , the present methods and compositions utilize substitution via homologous recombination into the glna1 locus , allowing selection based on a requirement for glutamine for growth . finally , substitution via homologous recombination into the glna1 locus with the use of a specific promoter allows high expression . selection of a strong promoter : as overexpression of the m . tuberculosis 30 kda major secretory protein from the chromosome would likely require a very strong promoter , the relative strength of several reportedly strong promoters was assessed using a plasmid system . the plasmid pre1 was first constructed from pnbv1 ( howard et al ., gene 166 : 181 - 182 , 1995 ) and a synthetic sequence generated by pcr assembly , which contained several strong transcriptional terminators ( t tonb , t fd , rrnbt 1 t 2 , t7te , and t teta ) to allow for the isolation of one or more transcriptional units . the m . tuberculosis glna1 gene was cloned into pre1 without a promoter or under the control of various promoters , and the resulting plasmids transformed by electroporation into an m . smegmatis glna1 mutant ( glutamine auxotroph ). the promoters selected for these studies were : p gyr , the promoter for the mycobacterium smegmatis dna gyrase genes ; p rrs , the promoter for the m . tuberculosis rrs gene ( also known as rrns , mtb000019 and the 16s ribosomal rna gene ); p rrs - short , a shortened derivative of p rrs lacking the boxa , boxb , and boxc elements ; p glna1 , the promoter for the m . tuberculosis glna1 gene , the glutamine synthetase glna1 protein gene ; p hsp60 , the promoter for the m . tuberculosis heat shock protein 60 gene also known as groel2 ; and p pknh , the promoter for the m . tuberculosis pknh gene , also known as rv1266c . the plasmid containing the glna1 gene without a promoter did not complement the glna1 mutant , indicating that background expression of glna1 ( due to read - through transcription from other promoters on the plasmid ) was tightly controlled . all of the plasmids containing the glna1 gene with a promoter were capable of complementing the glna1 mutant . cell lysates ( from equal volumes of culture ) of the m . smegmatis glna1 recombinant strains were analyzed by polyacrylamide gel electrophoresis and immunoblotting with polyvalent , highly specific rabbit anti - glna1 immunoglobulin ( fig1 a ). surprisingly , expression from the full length rrs promoter ( pre2 . 1 ) and the pknh promoter ( pre2 . 5 ) was relatively weak , in contrast to previous reports on these promoters ( agarwal and tyagi , fems microbiol lett 225 : 75 - 83 , 2003 ; verma et al ., gene 148 : 113 - 118 , 1994 ). expression from the p glna1 and p hsp60 promoters ( pre2 . 3 and pre2 . 4 , respectively ) was high , as we have previously observed for these promoters in other contexts ( tullius et al . infect immun 69 : 6348 - 6363 , 2001 ), however , the highest expression out of all the promoters was obtained with a shortened derivative of the rrs promoter ( p rrs - short , plasmid pre2 . 2 ) that was engineered to remove the boxa , boxb , and boxc sequences and incorporate a shine - dalgarno ( sd ) sequence matched to the m . tuberulosis 16s rrna anti - sd sequence . similar results were obtained when the same plasmids were used to transform a bcg glna1 mutant ( fig1 b ). the m . tuberculosis glna1 gene in pre2 . 2 was replaced with the m . tuberculosis 30 kda major secretory protein gene ( fbpb ) to generate plasmid pre4 . 2 . this plasmid was transformed into bcg by electroporation , and expression of the m . tuberculosis 30 kda major secretory protein ( fbpb ) from the p rrs - short promoter was very high , as observed for expression of glna1 from this promoter ( fig1 c ). unmarked integration of an expression cassette by a two step allelic exchange procedure : recombinant bcg strains that over - express and secrete the m . tuberculosis 30 kda major secretory protein were constructed by a two - step procedure that resulted in the stable integration of the fbpb gene into the bcg chromosome without leaving an antibiotic resistance marker , or any other extraneous dna , in the strains . in the first step , a bcg glna1 mutant ( glutamine auxotroph ) was generated from the parental bcg strain , via allelic exchange , incorporating a hygromycin resistance gene into the chromosome . in the second step , the fbpb gene along with a strong promoter was integrated into the glna1 locus , via allelic exchange , and at the same time the mutated glna1 allele was replaced with the wild - type glna1 allele . thus , the glutamine auxotroph from the first step was converted back to a glutamine prototroph and the hygromycin resistance gene was removed from the strain . for the first step , the allelic exchange substrate was generated using a cloning strategy in which a glna1 locus with a 852 by deletion at the 3 ′ end of the glna1 coding region was created with a hygromycin resistance ( hyg r ) gene inserted at the site of the deletion ( fig2 ; δglna1 :: hyg ). this mutated allele was cloned into the allelic exchange vector phex2 [ a derivative of phex1 , itself a derivative of phae87 ( bardarov et al ., microbiology 148 : 3007 - 3017 , 2002 )] to generate phex2 δglna1 :: hyg . this plasmid was electroporated into m . smegmatis to generate specialized transducing phage . bcg strains were infected with this purified phage and clones that had undergone a homologous recombination event were selected based on their resistance to hygromycin and then screened for glutamine auxotrophy . the allelic exchange substrates for the second step were generated using a cloning strategy in which an fbpb cassette , containing the fbpb gene with a strong promoter upstream to drive expression was cloned into the asci site of a wild - type glna1 locus , just downstream of the glna1 coding region ( fig2 ; 11a , 11b , 11c , 11d , 11e , 11f , and 13a ). these mutated alleles were cloned into the allelic exchange vector phex2 and specialized transducing phage was prepared in m . smegmatis , as above . bcg glutamine auxotrophs generated in the first step were infected with purified phage and clones that had undergone a homologous recombination event were selected based on their ability to grow in the absence of l - glutamine ( i . e . a functional glna1 allele was restored ). removal of the hygromycin gene was confirmed by culturing the strains on agar plates with and without hygromycin . hygromycin sensitive , glutamine prototrophs were screened for expression and export of recombinant m . tuberculosis 30 kda major secretory protein by polyacrylamide gel electrophoresis . this method was highly successful as 102 out of 111 clones that grew in the absence of l - glutamine were hygromycin sensitive ( 92 %) and 24 out of 25 of the hygromycin sensitive clones overexpressed the 30 kda major secretory protein ( 96 %). rbcg30 - armf - i tice and rbcg30 - armf - ii tice : the initial fbpb integration strains were constructed using rbcg ( pancd ) and rbcg ( mbtb ) as the parental strains . although expression of fbpb from the p rrs - short promoter was very strong on the plasmid pre4 . 2 ( fig1 c ), whether expression of fbpb would be affected once integrated into the chromosome or whether this expression might have a detrimental effect on the expression of the genes flanking the integration site , glna1 and glne , was unknown . therefore , six different fbpb integration strains were constructed using the fbpb cassette from pre4 . 2 that differed in the orientation of the fbpb gene and in the number of transcriptional terminators upstream and / or downstream of fbpb ( fig2 ; 11a , 11b , 11c , 11d , 11e , and 11f ). five of the six desired strains were constructed in the rbcg ( pancd ) parental strain ( no clones were obtained for 11c ) and the strains were analyzed for expression and export of recombinant m . tuberculosis 30 kda major secretory protein by polyacrylamide gel electrophoresis ( fig3 ). all five strains overexpressed the 30 kda major secretory protein , but the 11d , 11e , and 11f clones all expressed the protein at higher levels than the 11a and 11b clones . the 11a and 11b strains both contained three transcriptional terminators upstream of the p rrs - short promoter , while the other three strains lack these terminators . as orientation of fbpb in the 11d , 11e , and 11f strains did not affect expression , the lowered expression in the 11a and 11b strains is likely not due to blocking read - through transcription from upstream promoters , but due to down - regulating the strength of the p rrs - short promoter ( most likely due to the proximity of the rrnbt 1 t 2 terminator ). for comparison , strain 13a , which is similar to 11e except that the endogenous fbpb promoter was used instead of the p rrs - short promoter , was constructed . expression of the 30 kda major secretory protein was weak compared with strain 11e ( fig4 ). two strains were selected for further analysis ; a moderate expressing strain ( 11a ) and a high expressing strain ( 11e ). both strains stably expressed and exported the 30 kda major secretory protein for at least 28 generations ( 4 subcultures , 1 : 100 dilutions ). as these initial fbpb integration strains were constructed using rbcg ( pancd ) as the parental strain , the pancd locus needed to be restored to generate wild - type , prototrophic , fbpb integration strains that are completely free of antibiotic resistance markers . to accomplish this , an allelic exchange substrate encoding a wild - type pancd locus was cloned into the allelic exchange vector phex2 and specialized transducing phage was prepared in m . smegmatis , as above . the 11a and 11e strains were infected with purified phage and clones that had undergone a homologous recombination event were selected based on their ability to grow in the absence of pantothenate ( i . e . a functional pancd locus was restored ). removal of the apramycin gene that marked the pancd mutation was confirmed by culturing the strains on agar plates with and without apramycin . five apramycin sensitive , pantothenate prototrophs , for both the 11a and 11e strains , were randomly selected and screened for expression and export of recombinant m . tuberculosis 30 kda major secretory protein by polyacrylamide gel electrophoresis . all ten clones maintained a similar level of expression of the 30 kda major secretory protein compared with their parental strains ( 11a and 11e ), further evidence of the stability of the strains &# 39 ; expression levels ( fig5 ). a single clone derived from the 11a strain was selected and designated as rbcg30 - armf - i . likewise , a single clone derived from the 11e strain was selected and designated as rbcg30 - armf - ii . these two strains were compared to bcg and rbcg30 for expression and export of recombinant m . tuberculosis 30 kda major secretory protein by polyacrylamide gel electrophoresis ( fig6 ). the rbcg30 - armf - i strain was found to produce 9 . 5 fold more , and the rbcg30 - armf - ii strain was found to produce 15 . 5 fold more , of the 30 kda antigen per ml of culture than the control bcg tice strain . surprisingly , this expression of the 30 kda antigen from the chromosome was 1 . 6 fold and 2 . 6 fold more than that of rbcg30 where expression is from a multicopy plasmid . construction of an antibiotic resistance marker free plasmid containing an expression cassette and glna1 for balanced - lethal plasmid stabilization in mycobacterial glna1 strains : the plasmid pnbv1 - mtbgs , which was previously used to complement mycobacterial glna1 mutants , was first modified to contain an fbpb expression cassette that consists of the fbpb coding region along with the endogenous fbpb promoter , generating plasmids pnbv1 - mtbgs - fbpb - i and pnbv1 - mtbgs - fbpb - ii ( fig7 ). in plasmid pnbv1 - mtbgs - fbpb - i the fbpb gene is in the opposite orientation of glna1 and in plasmid pnbv1 - mtbgs - fbpb - ii the fbpb gene is in the same orientation as glna1 . the hygromycin resistance gene as well as the e . coli plasmid origin of replication were removed from these plasmids by digestion with clai and mfei , blunting of the dna termini , and self - ligation to recircularize the plasmids . the ligation products were electroporated into a m . smegmatis glna1 mutant and clones were selected that grew in the absence of glutamine ( i . e . the mutant was complemented by the plasmid ) and were hygromycin sensitive . total dna was isolated and plasmid dna was purified away from genomic dna on low melting point agarose gels . the plasmid dna was confirmed to be correct by restriction analysis and the plasmids were designated pnbv1 - mtbgs - fbpb - i δhyg and pnbv1 - mtbgs - fbpb - ii δhyg ( fig7 ). bcg glna1 pnbv1 - mtbgs - fbpb - i δhyg and bcg glna1 pnbv1 - mtbgs - fbpb - ii δhyg : the two plasmids described above lacking the hygromycin resistance gene and the e . coli plasmid origin of replication ( pnbv1 - mtbgs - fbpb - i δhyg and pnbv1 - mtbgs - fbpb - ii δhyg ) were electroporated into a bcg glna1 mutant and four clones ( for each plasmid ) that grew in the absence of glutamine ( i . e . the mutant was complemented by the plasmid ) were selected for expression analysis . all eight clones appeared to be overexpressing the 30 kda antigen . one clone of each strain was saved and designated bcg glna1 pnbv1 - mtbgs - fbpb - 1 δhyg and bcg glna1 pnbv1 - mtbgs - fbpb - ii δhyg . as the orientation of the fbpb expression cassette did not influence the expression level of the 30 kda antigen , one strain ( bcg glna1 pnbv1 - mtbgs - fbpb - i δhyg ) was selected for further analysis and was compared to bcg and other recombinant bcg strains overexpressing the 30 kda antigen ( fig8 ). bcg glna1 pnbv1 - mtbgs - fbpb - i δhyg had an expression profile similar to other recombinant bcg strains overexpressing the 30 kda antigen and was found to produce 10 . 6 fold more of the 30 kda antigen per ml of culture than the control bcg tice strain . in this particular example , the bcg glna1 mutant used to create the bcg glna1 pnbv1 - mtbgs - fbpb - i δhyg strain contains an antibiotic resistance marker in the chromosome ( a kanamycin resistance gene inserted in the glna1 gene ). the unmarked plasmids , pnbv1 - mtbgs - fbpb - i δhyg and pnbv1 - mtbgs - fbpb - ii δhyg , can be used in exactly the same way with an unmarked bcg glna1 strain to generate a vaccine strain that contains no antibiotic resistance markers . in one embodiment , immunogenic compositions are provided comprising a rbcg wherein the rbcg is metabolically impaired and wherein a siderophore and iron are used to regulate growth of the metabolically impaired strain . this rbcg has been rendered siderophore - dependent and iron - loadable . it can be grown in vitro in the presence of iron and a siderophore such as , but not limited to , mycobactin j or exochelin , and thereby loaded with iron . subsequently , when administered to the host , it can use the stored iron to multiply for several generations . as some growth of a live vaccine in the host is necessary to induce a strong protective immune response , the capacity of the rbcg to divide several times in the host allows the generation of a strong protective immune response . at the same time , the limited capacity of the rbcg to multiply in the host , as a result of its inability to acquire iron in the host , renders it unable to cause disseminated disease in the immunocompromised host and therefore safer than bcg . in another embodiment , growth regulatable recombinant bcg immunogenic compositions , which can not grow more than a few generations in the host without a nutritional supplement , are designed to be safer than bcg , because unlike bcg , such immunogenic composition can not disseminate in the host in the absence of the nutritional supplement . growth - regulatable recombinant bcg immunogenic compositions having antibiotic resistance markers are disclosed in co - pending international patent application pct / us2007 / 066348 , which is incorporated by reference herein for all it contains regarding growth regulatable recombinant bcg . growth - regulatable auxotrophic recombinant bcg immunogenic compositions are provided that are dependent upon small amounts of the vitamin pantothenate . the rbcg can be administered to the host without providing a nutrient supplement to the host , in which case it can only undergo a limited number of divisions using stored nutrient but a sufficient number of divisions to generate a potent protective immune response . alternatively , the vaccine can be administered to the host and the host provided a large amount of the nutrient , which can be given safely and inexpensively to mammals in large quantities , facilitating its acquisition by the live recombinant immunogenic composition in the host . in a non - limiting embodiment , the nutrient is the vitamin pantothenate . under such circumstances , the immunogenic composition can persist longer in the host and induce a stronger protective immune response . should the vaccine begin to disseminate and cause illness the nutrient supplement can be readily terminated , thereby stopping growth of the organism in the host and preventing serious disease . the amount of pantothenate normally present in the host eating a normal diet is orders of magnitude less than that needed to provide sufficient pantothenate for the growth of the rbcg . one embodiment of the live recombinant pantothenate - dependent bcg immunogenic composition over - expresses the m . tuberculosis 30 kda major secretory protein . embodiments therefore provide recombinant strains of bcg that are growth - limited and / or growth - regulatable including strains that secrete pathogen major extracellular proteins including m . tuberculosis major extracellular proteins . the immunogenic compositions are administered intradermally or by another route , e . g . subcutaneously , intranasally , inhaled , or even orally to a mammalian host . the immunogenic compositions are suitable for both immunocompetent and immunocompromised hosts . the immunogenic compositions induce a strong cell - mediated immune response to pathogen antigens in the vaccine . the immunogenic compositions subsequently protect the mammalian hosts against infection with m . tuberculosis , mycobacterium leprae , mycobacterium avium , other mycobacteria , and other intracellular pathogens . additionally , the current commercially available bcg vaccine against tuberculosis is of limited efficacy against pulmonary tuberculosis . the immunogenic compositions disclosed herein are more potent than the current commercially available vaccine in protecting against pulmonary tuberculosis and dissemination of bacteria to the spleen and other organs . additionally , the immunogenic compositions are safer than the current commercially available vaccine in that the immunogenic compositions are unable to disseminate in the immunocompromised host . despite the stability advantages of chromosome integration , expression of a recombinant antigen from a plasmid may produce a strain with a different phenotype than a strain expressing a recombinant antigen from the chromosome and therefore may potentially produce a superior immune response . therefore , the present disclosure allows for the expression of the desired antigen from a plasmid using balanced - lethal plasmid stabilization . the plasmid lacks antibiotic resistance markers and contains glna1 , which allows the plasmid to be stably maintained in mycobacterial glna1 mutants . previously , it was known that the immunostimulatory cytokines interleukin 2 ( il - 2 ), interleukin 12 ( il - 12 ), granulocyte - macrophage colony stimulating factor ( gm - csf ) and interferon gamma ( infγ ) are associated with enhanced cell - mediated immunity against intracellular pathogens including mycobacterium tuberculosis . for example , il - 12 enhances the resistance of mice to m . tuberculosis and mice lacking infγ show increased susceptibility to m . tuberculosis . these immunostimulatory cytokines , when present in close proximity to the m . tuberculosis 30 kda major secretory protein or other m . tuberculosis major extracellular proteins can enhance the protective immune response against tuberculosis induced by the extracellular proteins . moreover , a recombinant bcg immunogenic composition co - expressing one of these immunostimulatory cytokines and the 30 kda major secretory protein or other m . tuberculosis major extracellular proteins induces greater protective immunity than a recombinant bcg vaccine expressing the extracellular protein in the absence of the immunostimulatory protein . recombinant bcg immunogenic compositions expressing immunostimulatory proteins and having antibiotic resistance markers are disclosed in co - pending international patent application pct / us2007 / 066350 which is incorporated by reference herein for all it contains regarding immunostimulatory recombinant bcg . previous studies have shown that immunostimulatory cytokines , e . g . il - 2 and il - 12 , can augment the efficiency of subunit vaccines . however , none of the previously reported subunit vaccines have approached the efficacy of bcg . furthermore , previously disclosed cytokine - producing recombinant bcg vaccines did not induce more potent protection in animal models than rbcg alone . the present inventors have determined that a recombinant bcg vaccine expressing only infγ was not more potent than the parent bcg strain . surprisingly , the recombinant bcg co - expressing infγ and the 30 kda m . tuberculosis major secretory protein was more potent than rbcg30 , the strain only expressing the 30 kda protein . thus , when expressed by bcg , infγ did not enhance the level of protective immunity conferred by bcg alone , but when expressed by rbcg30 , it did enhance the level of protective immunity conferred by rbcg30 alone . therefore , the present inventors have determined that the co - expression of a majorly abundant extracellular antigen from an intracellular pathogen and a cytokine will result in enhanced protective immunity . the present disclosure provides recombinant bcg immunogenic compositions expressing cytokines including , but not limited to , interleukin - 2 ( il - 2 ), interleukin - 10 ( il - 10 ), interleukin - 12 ( il - 12 ), interleukin - 4 ( il - 4 ), interleukin - 6 ( il - 6 ), interleukin - 7 ( il - 7 ), interleukin - 8 ( il - 8 ), interleukin - 15 ( il - 15 ), interleukin - 18 ( il - 18 ), interferon gamma , tumor necrosis factor alpha ( tnf - alpha ), granulocyte macrophage colony stimulating factor ( gm - csf ). the human cytokines il - 2 , il - 12 , and gm - csf have been reported to be active in the guinea pig and active in non - glycosylated form . additionally , rbcgs expressing cytokine receptors such as , but not limited to , the soluble il - 4 receptor ( sil4r ) and the receptors for il - 2 , il - 4 , il - 7 , il - 12 , ifns , gm - csf or tnf - alpha are disclosed . the studies of the efficacy of the vaccines utilized guinea pigs because the guinea pig model is especially relevant to human tuberculosis clinically , immunologically , and pathologically . in contrast to the mouse and rat , but like the human , the guinea pig a ) is susceptible to low doses of aerosolized m . tuberculosis ; b ) exhibits strong cutaneous delayed - type hypersensitivity ( dth ) to tuberculin ; and c ) displays langhans giant cells and caseation in pulmonary lesions . however , whereas only about 10 % of immunocompetent humans who are infected with m . tuberculosis develop active disease over their lifetime ( half early after exposure and half after a period of latency ), infected guinea pigs always develop early active disease . while guinea pigs differ from humans in this respect , the consistency with which they develop active disease after infection with m . tuberculosis is an advantage in trials of vaccine efficacy . aliquots were removed from logarithmically growing wild - type or recombinant bcg cultures , and the bacteria were pelleted by centrifugation at 3 , 500 × g for 15 min . the bacteria are then washed with 1 × phosphate buffered saline ( 1 × pbs , 50 mm sodium phosphate ph 7 , 150 mm sodium chloride ) and resuspended at a final concentration of 1 × 10 4 or 1 × 10 7 colony - forming units per ml in 1 × pbs . the immunization inoculum contains 10 3 or 10 6 viable wild - type or recombinant bcg bacteria in a total volume of 100 μl . specific - pathogen free 250 - 300 g outbred male hartley strain guinea pigs from charles river breeding laboratories , in groups of 15 or 21 , were sham - immunized by intradermal administration of buffer ( 15 animals total ) or immunized intradermally with 10 3 cfu of one of the following strains of recombinant bcg ( 21 animals / group ): cutaneous delayed - type hypersensitivity ( dth ) to purified recombinant m . tuberculosis 30 kda major secretory protein ( r30 ) ten weeks after immunization , 6 guinea pigs in each group were shaved over the back and injected intradermally with 10 μg of purified recombinant m . tuberculosis 30 kda major secretory protein ( r30 ) in 100 μl phosphate buffered saline . after 24 h , the diameter of erythema and induration was measured . a separate group of animals from the one used in the challenge studies is used for skin - testing to eliminate the possibility that the skin test itself might influence the outcome . the results are summarized in table 1 . these results showed that sham - immunized animals ( group a ) and animals immunized with the parental bcg tice strain ( group b ) had no induration upon testing with r30 . animals immunized with the unmarked strain rbcg30 - armf - i ( group j ), had little induration upon testing with r30 . in contrast , animals immunized with the unmarked strain rbcg30 - armf - ii ( group k ) had significant induration upon testing with r30 , similar to animals immunized with rbcg30 ( group c ). ten weeks after immunization , the remaining animals were challenged with an aerosol generated from a 10 ml single - cell suspension containing 3 × 10 4 colony forming units ( cfu ) of m . tuberculosis per ml . prior to challenge , the challenge strain , m . tuberculosis erdman strain ( atcc 35801 ), was passaged through outbred guinea pigs to maintain virulence , cultured on 7h11 agar , subjected to gentle sonication to obtain a single cell suspension , and frozen at − 70 ° c . this aerosol dose delivers approximately 10 live bacilli to the lungs of each animal . the airborne route of infection was used because this is the natural route of infection for pulmonary tuberculosis . a relatively large dose was used so as to induce measurable clinical illness in 100 % of control animals within a relatively short time frame ( 10 weeks ). afterwards , guinea pigs were individually housed in stainless steel cages contained within a laminar flow biohazard safety enclosure and allowed free access to standard laboratory chow and water . the animals were observed for illness and weighed weekly for 10 weeks and then euthanized . the right lung and spleen of each animal was removed and cultured for cfu of m . tuberculosis on middlebrook 7h11 agar for two weeks at 37 ° c ., 5 % co 2 - 95 % air atmosphere . the results of the assay for cfu in the lungs and spleens are shown in table 2 . these results showed that animals immunized with bcg or any recombinant bcg strain had much lower cfu in the lungs and spleens than the sham - immunized animals . animals immunized with the unmarked strain rbcg30 - armf - i ( group j ) had fewer cfu in the lung ( 0 . 15 log ) and spleen ( 0 . 32 log ) than bcg ( group b ), however the differences were not statistically significant . in contrast , animals immunized with the unmarked strain rbcg30 - armf - ii ( group k ) had even fewer cfu in the lung ( 0 . 42 log , p = 0 . 01 by anova ) and spleen ( 0 . 63 log , p = 0 . 002 by anova ) compared with bcg and the differences were statistically significant . antibody to purified recombinant m . tuberculosis 30 kda major protein ( r30 ) blood is obtained from the animals described above immediately after they are euthanized , and the serum is assayed for antibody titer to r30 by elisa , using costar ( corning , n . y .) 96 - well eia / ria high binding plates , r30 at 1 μg / well , guinea pig serum diluted 1 : 64 to 1 : 1 , 024 , 000 , alkaline phosphatase - conjugated goat anti - guinea pig igg ( sigma , st . louis , mo .) at a dilution of 1 : 1 , 000 , and an alkaline phosphatase substrate kit ( biorad , hercules , calif .). three weeks after immunization , animals are euthanized and the spleen removed for lymphocyte proliferation studies . splenic lymphocytes are purified as described ( pal and horwitz , infect . immun . 60 : 4781 - 4792 , 1992 ) and incubated at a final concentration of 10 7 / ml in rpmi1640 containing 12 . 5 mm hepes , penicillin ( 100 u / ml ), streptomycin ( 100 μg / ml ), polymyxin b sulfate ( 100 units / ml ), and 10 % fetal calf serum ( gibco ) with ppd ( 10 mg / ml ) or with 100 , 10 , or 1 μg / ml of purified m . tuberculosis 30 kda major secretory protein ( r30 ) in a total volume of 100 μl in microtest wells ( 96 - well round - bottom tissue culture plate ; falcon labware , oxnard , calif .) for 2 days at 37 ° c . in 5 % co 2 - 95 % air and 100 % humidity . as negative and positive controls , lymphocytes sre incubated with buffer only ( rpmi ) or with concanavalin a ( 15 μg / ml ). subsequently , [ 3 h ] thymidine incorporation is determined and mean counts per minute ( cpm ) calculated . stimulation indices ( si ) are calculated using the following formula : si = cpm with antigen / cpm without antigen . lymphocytes from animals immunized with bcg have a weak proliferative response to r30 , but a moderately strong response to ppd . in contrast , lymphocytes from animals immunized with rbcg expressing the 30 kda protein have a strong proliferative response to both r30 and ppd . agarwal , n ., and tyagi , a . k . 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( 1994 ). functional analysis of transcription of the mycobacterium tuberculosis 16s rdna - encoding gene . gene 148 , 113 - 118 . unless otherwise indicated , all numbers expressing quantities of ingredients , properties such as molecular weight , reaction conditions , and so forth used in the specification and claims are to be understood as being modified in all instances by the term “ about .” accordingly , unless indicated to the contrary , the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention . at the very least , and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims , each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques . notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations , the numerical values set forth in the specific examples are reported as precisely as possible . any numerical value , however , inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements . the terms “ a ,” “ an ,” “ the ” and similar referents used in the context of describing the invention ( especially in the context of the following claims ) are to be construed to cover both the singular and the plural , unless otherwise indicated herein or clearly contradicted by context . recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range . unless otherwise indicated herein , each individual value is incorporated into the specification as if it were individually recited herein . all methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context . the use of any and all examples , or exemplary language ( e . g ., “ such as ”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed . no language in the specification should be construed as indicating any non - claimed element essential to the practice of the invention . groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations . each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein . it is anticipated that one or more members of a group may be included in , or deleted from , a group for reasons of convenience and / or patentability . when any such inclusion or deletion occurs , the specification is deemed to contain the group as modified thus fulfilling the written description of all markush groups used in the appended claims . certain embodiments of this invention are described herein , including the best mode known to the inventors for carrying out the invention . of course , variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description . the inventor expects skilled artisans to employ such variations as appropriate , and the inventors intend for the invention to be practiced otherwise than specifically described herein . accordingly , this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law . moreover , any combination of the above - described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context . furthermore , numerous references have been made to patents and printed publications throughout this specification . each of the above - cited references and printed publications are individually incorporated herein by reference in their entirety . in closing , it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention . other modifications that may be employed are within the scope of the invention . thus , by way of example , but not of limitation , alternative configurations of the present invention may be utilized in accordance with the teachings herein . accordingly , the present invention is not limited to that precisely as shown and described .