Patent Publication Number: US-7722861-B2

Title: Attenuated Mycobacterium tuberculosis vaccines

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Application No. 60/358,152, filed Feb. 19, 2002. That application is incorporated by reference herewith in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     The present invention was supported by NIH Grant No. AI26170. As such, the U.S. Government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     (1) Field of the Invention 
     The present invention generally relates to live bacterial vaccines. More specifically, the invention is related to novel  Mycobacterium sp.  compositions, and the use of those compositions to protect mammals against disease caused by virulent  Mycobacterium  sp. 
     (2) Description of the Related Art 
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     There exists an urgent need for a novel tuberculosis (TB) vaccine as there are more than 8 million new cases of tuberculosis and more than 2 million deaths reported each year by the WHO (Dye et al., 1999). The discovery of the causative agent of TB,  Mycobacterium tuberculosis , by Robert Koch in 1882 opened up the possibility for a novel vaccine (Koch, 1882). Since then, numerous attempts to develop attenuated vaccines against tuberculosis have failed, including tuberculin (a protein extract of killed  tubercle bacilli ) developed by Dr. Koch himself. This failure of tuberculin to protect led to a “firm conviction that immunity could only be established by inducing a definite, albeit limited, tuberculosis process” Grange et al., 1983). Thus, numerous labs set out to follow the example of Dr. Louis Pasteur for viruses and enrich attenuated mutants of the  tubercle bacillus  following repeated passaging. 
     In order to test the hypothesis that a  tubercle bacillus  isolated from cattle (now known as  M. bovis ) could transmit pulmonary tuberculosis following oral administration, Drs. Calmette and Guerin developed a medium containing beef bile that enabled the preparation of fine homogenous bacillary suspensions (Calmette and Guerin, 1905). An  M. bovis  strain obtained from Dr. Norcard, was passaged every 21 days in this medium and after the 39 th  passage, the strain was found to be unable to kill experimental animal (Gheorghiu, 1996). “Between 1908 and 1921, the strain showed no reversion to virulence after 230 passages on bile potato medium” (Id.), which is consistent with the attenuating mutation being a deletion mutation. In the animal studies that followed, the strain (‘BCG’) was found to be attenuated but it also protected animals receiving a lethal challenge of virulent  tubercle bacilli  (Calmette and Guerin, 1920). BCG was first used as a vaccine against tuberculosis in 1921. From 1921 to 1927, BCG was shown to have protective efficacy against TB in a study on children (Weill-Halle and Turpin, 1925; Calmette and Plotz, 1929) and adopted by the League of Nations in 1928 for widespread use in the prevention of tuberculosis. By the 1950&#39;s after a series of clinical trials, the WHO was encouraging widespread use of BCG vaccine throughout the world (Fine and Rodrigues, 1990). Although an estimated 3 billion doses have been used to vaccinate the human population against tuberculosis, the mechanism that causes BCG&#39;s attenuation remains unknown. 
     Mahairas et al. (1996) first compared the genomic sequences of BCG and  M. bovis  using subtractive hybridization and found that there were three major deletions (named RD1, RD2, and RD3) present in the genome of  M. bovis , but missing in BCG. Behr et al. (1999) and others (Gordon et al., 2001) later identified 16 large deletions, including RD1 to RD3, present in the BCG genome but absent in  M. tuberculosis . These authors concluded that 11 of these 16 deletions were unique to  M. bovis , while the remaining 5 deletions were unique to BCG. They also found that one of these 5 deletions, designated RD1 (9454 bp), is present in all of the BCG substrains currently used as TB vaccines worldwide and concluded that the deletion of RD1 appeared to have occurred very early during the development of BCG, probably prior to 1921 (Behr et al., 1999). 
     The development of insertional mutagenesis systems for BCG and  M. tuberculosis  (Kalpana et al., 1991), transposon mutagenesis systems (Cirillo et al., 1991; McAdam et al., 1995; Bardarov et al., 1997) and allelic exchange systems (Balasubramanian et al., 1996; Pelicic et al., 1997) led to the isolation of the first auxotrophic (nutrient-requiring) mutants of these slow-growing mycobacteria. Auxotrophic mutants of BCG and  M. tuberculosis  have been shown to confer protection to  M. tuberculosis  challenges with variable efficacies (Guleria et al., 1996; Smith et al., 2001). However, a head-to-head comparison of BCG to a leucine auxotroph of BCG showed that a single immunization elicited no positive skin-test and imparted little immunity to challenges with  M. tuberculosis  or  M. bovis  (Chambers et al., 2000). In contrast, a methionine auxotroph of BCG that grows in vivo did confer significant protection to challenge to both  M. tuberculosis  and  M. bovis  (Id.). A single dose of a leucine auxotroph of  M. tuberculosis  failed to elicit protection as good as BCG in BALB/c mice (Hondalus et al., 2000). These results suggest that optimal immunity against  M. tuberculosis  requires some growth of the immunizing strain. Double mutants of  M. tuberculosis  have also been created (Parish and Stoker, 2000), but whether such mutants are improved over single attenuating mutants in protecting mammals against challenge with a virulent  mycobacterium , particularly when the host is immunocompromised, has not been established. 
     It is also worth noting that in the study of Chambers et al. (2000), both BCG and the BCG mutants seemed to protect better against  M. bovis  challenge than  M. tuberculosis . If we assume the reverse correlate is true, we could hypothesize that optimal immunity against  M. tuberculosis  could be achieved with  M. tuberculosis -derived mutant that grew in the mammalian host. 
     Based on the above, there remains a need for improved live mycobacterial vaccines having attenuated virulence, that confer protection from virulent mycobacteria, particularly  M. tuberculosis . The instant invention satisfies that need. 
     SUMMARY OF THE INVENTION 
     The present invention is based on the discovery that deletion of the RD1 region or a region controlling the production of a vitamin from the genome of virulent mycobacteria in the  M. tuberculosis  complex attenuates the virulence of the mycobacteria without eliminating the ability of the mycobacteria to colonize susceptible mammals. These attenuated mycobacteria are capable of protecting the mammals from challenge by a virulent  M. tuberculosis  complex mycobacteria. The attenuated mycobacteria are thus useful in methods and compositions for vaccination of humans, cows and other mammals from virulent  M. tuberculosis  complex mycobacteria. 
     Accordingly, in some embodiments, the present invention is directed to a non-naturally occurring  Mycobacterium tuberculosis . The  M. tuberculosis  comprises a deletion of an RD1 region or a region controlling production of a vitamin. The  M. tuberculosis  preferably exhibits attenuated virulence in a mammal when compared to the  M. tuberculosis  without the deletion. 
     In certain aspects of these embodiments, the  Mycobacterium tuberculosis  is produced by deletion of an RD1 region or a region controlling production of a vitamin. In these aspects, the  M. tuberculosis  also preferably exhibits attenuated virulence in a mammal when compared to the  M. tuberculosis  without the deletion. 
     In related embodiments, the present invention is also directed to mycobacteria in the  M. tuberculosis  complex that are genetically engineered to comprise a deletion of an RD1 region or a region controlling production of a vitamin. 
     The present invention is also directed to mycobacteria in the  M. tuberculosis  complex that comprise a deletion of a region controlling production of a vitamin. These mycobacteria are preferably capable of sustaining an infection in an immunocompetent mouse for at least 20 weeks. 
     The inventors have also discovered that mycobacteria that are auxotrophic for lysine have attenuated virulence and can protect a mammal against challenge by a virulent  mycobacterium . Accordingly, the invention is also directed to non-naturally occurring mycobacteria in the  M. tuberculosis  complex, wherein the mycobacteria comprise a deletion of a region controlling production of lysine, and wherein the mycobacteria are capable of sustaining an infection in an immunocompetent mouse for at least 20 weeks. 
     The inventors have additionally discovered that mycobacteria having two attenuating deletions are highly attenuated, even in immunocompromised mammals, and are surprisingly effective in protecting mammals against challenge by a virulent microorganism. Thus, the invention is additionally directed to mycobacteria in the  M. tuberculosis  complex that are genetically engineered to comprise two deletions. The two deletions are any deletions where a virulent  mycobacterium  in the  M. tuberculosis  complex having either deletion exhibits attenuated virulence. 
     In further embodiments, the invention is directed to tuberculosis vaccines comprising any of the above-described  M. tuberculosis  or mycobacteria in the  M. tuberculosis  complex, in a pharmaceutically acceptable excipient. These vaccines are capable of protecting mammals from challenge by virulent mycobacteria in the  M. tuberculosis  complex. 
     The invention is also directed to methods of protecting mammals from virulent  M. tuberculosis  or mycobacteria in the  M. tuberculosis  complex. The methods comprise treating the mammal with any of the above vaccines. 
     In other embodiments, the invention is directed to methods of preparing tuberculosis vaccines. The methods comprise deleting an RD1 region or a region controlling production of a vitamin or lysine from an  M. tuberculosis  to produce any of the  M. tuberculosis  described above. In these embodiments, the vaccine is capable of protecting the mammal from challenge by a virulent  M. tuberculosis.    
     In related embodiments, the invention is directed to other methods of preparing a tuberculosis vaccine. These methods comprise genetically engineering a  mycobacterium  to delete an RD1 region or a region controlling production of a vitamin or lysine to produce any of the mycobacteria described above. In these embodiments, the vaccine is capable of protecting the mammal from challenge by a virulent mycobacteria of the  M. tuberculosis  complex. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows maps and autoradiographs pertaining to the construction of ΔRD1 mutants of  M. tuberculosis . Panel a,  M. tuberculosis  H37Rv published sequence between 4346 kb and 4364 kb, showing predicted NcoI sites. Arrows on the top represent the genes in the RD1 region. The RD1 region deleted from  M. bovis  BCG is represented by an open bar spanning from Rv3871 to Rv3879c. Upstream and downstream flanking sequences, UFS and DFS respectively, are indicated as closed bars underneath the grid line. Panel b, Southern hybridization of  M. tuberculosis  H37Rv ΔRD1 created using two-step sequential homologous recombination. Panel c, Southern hybridization of  M. tuberculosis  H37Rv and Erdman ΔRD1 strains created using specialized transduction. 
         FIG. 2  shows graphs summarizing experiments establishing that  M. tuberculosis  H37Rv ΔRD1 is attenuated in SCID mice. Panel a, Seven female SCID mice were infected intravenously with 2×10 6  CFU  M. tuberculosis  H37Rv,  M. tuberculosis  H37Rv ΔRD1, and  M. tuberculosis  H37Rv ΔRD1::2F9 per mouse. The number of surviving mice was recorded post infection. Panel b, Mice were infected with different doses of  M. tuberculosis  H37Rv,  M. tuberculosis  H37Rv ΔRD1, and  M. bovis  BCG. For each strain, infection doses of 2×10 6  CFU, 2×10 5  CFU, 2×10 4  CFU, and 2×10 3  CFU per mouse, were administered via tail intravenous injection. 
         FIG. 3  is photographs, micrographs and autoradiographs showing that the  M. tuberculosis  H37Rv ΔRD1 mutant exhibits two distinct colonial morphotypes. Panel a,  M. tuberculosis  H37Rv. Panel b,  M. tuberculosis  H37Rv ΔRD1. Panel c,  M. tuberculosis  H37Rv ΔRD1::2F9. Panel d, Southern analysis of  M. tuberculosis  H37Rv ΔRD1 NcoI-digested genomic DNA, isolated from three smooth and three rough colonies and probed with DFS. Panels e-g, Colonial morphotypes at higher magnification. e, Smooth morphotype at week 4. f, Rough morphotype at week 4. g, Rough morphotype at week 6. 
         FIG. 4  is graphs showing the growth kinetics of  M. tuberculosis  H37Rv ΔRD1 in BALB/c mice. Mice were infected with 2×10 6  CFU through tail injection. Time to death was noted and at day 1, week 4, 8, 14, and 22 post-infection, mice were sacrificed to determine the mycobacterial burden in the spleen, liver, and lung. The numbers represent the means of CFUs in organs derived from three animals. The error bars represent the standard errors of the means. Panel a, Time to death assay in BALB/c mice. Panel b, Spleen. Panel c, Liver. Panel d, Lung. 
         FIG. 5  is micrographs from pathological studies of infected BALB/c mice. Panels a-c, Lungs from mice infected with 2×10 6  CFU of  M. tuberculosis  H37Rv examined at 4, 8 and 14 weeks post-infection. The mild to moderate pneumonia at 4 and 8 weeks (a and b) progressed to severe consolidating granulomatous pneumonia at 14 weeks post infection (c). Panels d-f, Lungs from mice infected with 2×10 6  CFU of  M. tuberculosis  H37Rv ΔRD1 examined at 4, 8 and 22 weeks post-infection showing moderate pneumonia at 8 weeks post-infection (e) and persistent bronchitis and multifocal pneumonitis at 22 weeks post-infection (f). Panels (g)-(i), Mild lung lesions from mice infected with 2×10 6  CFU of BCG at 4, 8 and 22 weeks post-infection. Mild focal granulomas scattered widely in the lung at each time point with predominately lymphocytic accumulations in foci at 22 weeks post-infection. 
         FIG. 6  shows graphs summarizing experiments establishing that pantothenate auxotrophy leads to attenuation of  M. tuberculosis  ΔpanCD mutant in SCID mice. Panel A, Survival of BALB/c SCID mice (n=12 per group) infected intravenously with 490 CFU of H37Rv (∘) or 210 CFU of panCD complementing strain (panCD in single copy integrated into the chromosome)(●) or 3.4×10 5  CFU of ΔpanCD mutant (▴) or 3.3×10 5  CFU of BCG-P (□). Panel B, Bacterial numbers in the spleen (●) and lungs (▴) of SCID mice infected intravenously with 490 CFU of H37Rv or numbers in spleen (∘) and lungs (Δ) of mice infected with 3.4×10 5  CFU of ΔpanCD mutant. The results represent means± standard errors of four to five mice per group. 
         FIG. 7  shows graphs summarizing experiments demonstrating the attenuation, limited growth and persistence of ΔpanCD mutant in immunocompetent mice. Panel A, Survival of BALB/c mice (n=12 per group) infected with 4.4×10 6  CFU of wild-type  M. tuberculosis  H37Rv (∘), 3.2×10 6  CFU panCD complementing strain (panCD in single copy integrated into the chromosome)(●) or 2.4×10 6  CFU panCD mutant (▴). Panels B and C, Bacterial loads in spleen and lungs of BALB/c mice infected intravenously with 4.4×10 6  CFU wild-type H37Rv (∘) or 3.2×10 6  CFU panCD complementing strain (●) or 2.4×10 6  CFU ΔpanCD mutant (▴). CFUs were assayed at various time points on 7H11 agar with or without pantothenate supplementation where required. The results represent means± standard errors of four to five mice per group. 
         FIG. 8  shows graphs summarizing experiments demonstrating the attenuation, limited replication and persistence of ΔnadBC mutant in immunocompetent mice. Panels A and B, Bacterial loads in lungs and spleen of C57BL/6 mice infected with wild type  M. tuberculosis  H37Rv (●) or ΔnadBC mutant (∘). Mice were infected intravenously with 10 6  CFU of each strain. CFUs were assayed at various time points on 7H11 agar with or without nicotinamide supplementation where required. The results represent means± standard errors of four to five mice per group. Panel C, Survival of C57BL/6 mice (n=12 per group) infected with 10 6  CFU of wild-type bacteria (●) or 10 6  CFU of ΔnadBC mutant (∘). 
         FIG. 9  shows an illustration, map and autoradiograph relating to the pathway for the biosynthesis of pantothenic acid and coenzyme A and its disruption in  M. tuberculosis . Panel a. The enzymes involved in the biosynthesis of pantothenic acid and having annotation in the genomic sequence of  M. tuberculosis  H37Rv are shown in bold numbers: 1) panB, ketopantoate hydroxymethyl transferase; 2) panD, aspartate-1-decarboxylase; 3) panC, pantothenate synthetase; 4) panK, pantothenate kinase; 5) acpS, ACP synthase. Panel b. Map of the panCD genomic region in the wild type  M. tuberculosis  H37Rv and the ΔpanCD mutant. Restriction sites and probe location are indicated. Panel c. Southern blot of BssHII-digested genomic DNA from wild-type H37Rv (lane 1), two independent clones of ΔpanCD mutant from H37Rv (lanes 2 &amp; 3) and probed with a 716 bp downstream region flanking the panCD operon. Molecular size marker (in kb) is shown on the left. 
         FIG. 10  shows graphs summarizing experiments demonstrating that pantothenate auxotrophy leads to attenuation of ΔpanCD mutant in mice. a. Survival of BALB/c SCID mice (n=12 per group) infected intravenously with H37Rv (∘) or panCD-complemented strain (●) or ΔpanCD mutant (▴) or  M. bovis  BCG-P (□). b. Bacterial numbers in the spleen (∘), liver (□) and lung (Δ) of SCID mice infected intravenously with H37Rv or the bacterial numbers in the spleen (●), liver (▪) and lung (▴) of mice infected with ΔpanCD mutant. c. Survival of immunocompetent BALB/c mice (n=16 per group) infected with H37Rv (∘) or panCD-complemented strain (●) or ΔpanCD mutant (▴). d, e, f. Bacterial numbers in lung (d), spleen (e) and liver (f) of immunocompetent BALB/c mice infected intravenously with either H37Rv (∘), panCD-complemented strain (●) or ΔpanCD mutant (▴). Data are means± standard errors of four to five mice per group. 
         FIG. 11  shows micrographs (Panels a-d) and graphs (Panels e and f) summarizing experiments demonstrating that the ΔpanCD mutant produces less tissue pathology in lungs of infected BALB/c mice and protects mice against challenge with virulent  M. tuberculosis . Panel a. Severe consolidating granulomatous pneumonia (★) obliterating the normal lung parenchyma at 3 weeks post-infection with H37Rv. Panel b. Severe consolidating granulomatous pneumonia (★) obliterating the normal lung parenchyma at 3 weeks post-infection with the panCD-complemented strain, similar to the wild type strain. Panel c. Mild lung infection caused by the ΔpanCD mutant at 3 weeks post-infection. Localized multifocal granulomas (arrows) scattered widely in the lung. Most of the lung is normal alveolar spaces and airways. Panel d. Lung of mouse infected with ΔpanCD mutant examined histologically at 23 weeks post-infection. Occasional focal, mild perivascular and interstitial infiltrations composed of predominately lymphocytes (arrows). Most of the lung is normal alveolar spaces and airways. e, f. The attenuated ΔpanCD mutant protects mice against aerogenic challenge with virulent  M. tuberculosis  Erdman. Subcutaneously immunized mice were challenged after 90 days through the aerosol route. The CFU numbers reflect the bacterial burden at 28 days post aerosol challenge in the lung (e) and spleen (f). Naive mice—black fill; mice infected with 1 dose panCD—light shade; mice infected with 2 doses panCD—dark shade; mice infected with BCG-P—unshaded. 
         FIG. 12  shows autoradiographs of Southern analysis of the NcoI-digested genomic DNA isolated from the wild type and the ΔRD1 mutants generated using specialized transduction in  M. tuberculosis  and  M. bovis . Lanes: 1— M. tuberculosis  H37Rv; 2— M. tuberculosis  H37Rv ΔRD1; 3— M. tuberculosis  Erdman; 4— M. tuberculosis  Erdman ΔRD1; 5— M. tuberculosis  CDC1551; 6— M. tuberculosis  CDC1551 ΔRD1; 7— M. bovis  Ravenel; and 8— M. bovis  Ravenel ΔRD1. The probes used in the Southern analysis was either DFS (left) or IS6110-specific sequence (right). 
         FIG. 13  shows graphs summarizing data confirming that deletion of RD1 in  M. tuberculosis  and  M. bovis  confers an attenuation of virulence for  M. tuberculosis  and  M. bovis , as indicated by these Time to death curves of mice infected intravenously with 2×10 6  CFU mycobacteria. Panel A, SCID mice infected with  M. tuberculosis  H37Rv (▪),  M. tuberculosis  H37Rv ΔRD1 (□),  M. tuberculosis  Erdman (●),  M. tuberculosis  Erdman ΔRD1 (∘),  M. tuberculosis  CDC1551 (▴),  M. tuberculosis  CDC1551 ΔRD1 (Δ),  M. bovis  Ravenel (▾),  M. bovis  Ravenel ΔRD1 (∇); Panel B, SCID mice infected intravenously with  M. tuberculosis  H37Rv (●),  M. tuberculosis  ΔRD1 (▪),  M. tuberculosis  ΔRD1::2F9 (▴),  M. bovis  Ravenel (∘),  M. bovis  Ravenel ΔRD1 (□), and  M. bovis  BCG (Δ); Panel C, BALB/c mice were infected with  M. tuberculosis  H37Rv (∘),  M. tuberculosis  ΔRD1 (Δ), and  M. bovis  BCG (□). 
         FIG. 14  are graphs summarizing experiments demonstrating the clearance of the lysine auxotroph in SCID mice. The viable bacterial counts are shown for the spleens, livers, and lungs of SCID mice injected intravenously with the lysine auxotroph strain and the prototrophic control strain. Three mice were assayed at each time point. The error bars indicate the standard deviations of the mean values. Note that the counts at time zero are the counts obtained at 24 hours post-injection, as described in Example 5. Panels A, B and C show the log of the viable bacteria in each organ after injection with 1×10 7  CFU of the Lys −    M. tuberculosis  mutant mc 2 3026 (□), or 1×10 7  CFU of the complemented Lys +    M. tuberculosis  strain mc 2 3026/pYUB651 (▪). 
         FIG. 15  is graphs summarizing experimental results of experiments that establish the vaccine efficacy of the  M. tuberculosis  lysine auxotroph mc 2 3026. C57B1/6 mice were injected intravenously with 1×10 6  CFU of the  M. tuberculosis  lysine auxotroph mc 2 3026, followed by one or two additional injections at 4 week intervals. Five mice were sacrificed weekly after each immunization and the viable bacteria counts of the auxotroph determined in the lungs and spleens. Control mice were given a similar amount of BCG-Pasteur or only PBST. Shown in Panel A is the clearance of the auxotroph from the lungs of the mice after each immunization period; one injection (▪), two injections (♦), and three injections (●). Three months after the initial immunization the vaccinated and control mice were challenged with virulent  M. tuberculosis  Erdman by the aerosol route. Five challenge mice were sacrificed following the challenge period and the lung homogenates plated to check the viable counts of the challenge inoculum. Groups of vaccinated and control mice were sacrificed at 14, 28, and 42 days later and the lung and spleen homogenates plated to determine viable colony forming units. Shown in Panel B are the viable challenge bacteria per lung of mice given one dose of the  M. tuberculosis  lysine auxotroph, and in panel C, the viable challenge bacteria per lung of mice given two doses of the auxotroph. Key: Viable challenge bacteria per lung of mice given the  M. tuberculosis  lysine auxotroph mc 2 3026 (▪), BCG-Pasteur (⋄), or PBST (∘). P values are indicated in the figure. Note that the results shown here are for the lungs. Similar results (not shown) were obtained from the spleens in all the experiments. 
         FIG. 16  shows a graph summarizing experiments establishing the survival curves of mice immunized three times with the  M. tuberculosis  lysine auxotroph mc 2 3026. C57B1/6 mice were injected intravenously with 1×10 6  CFU of the  M. tuberculosis  lysine auxotroph mc 2 3026, followed by two more injections at 4 week intervals, and challenged as described in Example 5. The percent survival is shown for mice immunized thrice with the  M. tuberculosis  lysine auxotroph mc 2 3026 (▪, 5 mice total), once with BCG-Pasteur (♦, 5 mice), and for the PBST controls (●, 10 mice). 
         FIG. 17  shows graphs summarizing experimental results establishing that the virulence of strain mc 2 6030 is highly attenuated in SCID mice and BALB/c mice. 
         FIG. 18  shows graphs summarizing experimental results measuring growth of various strains of  M. tuberculosis  in spleen (Panel A) and lungs (Panel B) of C57BL/6 mice. 
         FIG. 19  is a graph summarizing experimental results establishing that immunization with mc 2 6020 and mc 2 6030 protects mice against TB as effectively as BCG. This graph shows the survival of C57BI/6 mice challenged with virulent  M. tuberculosis  Erdman through the aerosol route three months after a single dose subcutaneous immunization with either BCG, mc 2 6020 (ΔlysAΔpanCD) or mc 2 6030 (ΔRD1ΔpanCD) and compared to non-immunized naive mice. There were 12 to 15 mice in each survival group. 
         FIG. 20  shows graphs summarizing experimental results establishing that  M. tuberculosis  double deletion mutants are highly attenuated in SCID mice. A dose of 10 5  mc 2 6020 or mc 2 6030 were intravenously inoculated into SCID mice (10 per group) and time to death assessments were performed. While the same dose of  M. tuberculosis  and BCG killed mice in 40 or 90 days, respectively, the mice infected with mc 2 6020 or mc 2 6030 survived over 400 or 250 days, respectively. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is based in part on the discovery that virulent mycobacteria in the  M. tuberculosis  complex that have deletions in the RD1 region, or in a region that controls production of a vitamin, are attenuated in virulence but are capable of sustaining viability and growth in a mammalian host, and are also capable of protecting against a challenge by a virulent  M. tuberculosis  complex  mycobacterium.    
     Thus, in some embodiments, the invention is directed to non-naturally occurring  Mycobacterium tuberculosis  that comprise a deletion of an RD1 region or a region controlling production of a vitamin. These  M. tuberculosis  preferably exhibit attenuated virulence in a mammal when compared to the  M. tuberculosis  without the deletion. 
     A host organism can be inoculated with the mycobacteria of the present invention by any of a number of ways known in the art. These include oral ingestion, gastric intubation, or broncho-nasal-ocular spraying. Other methods of administration include intravenous, intramuscular, intramammary, or, preferably, subcutaneous or intradermal injection. The immunization dosages required can be determined without undue experimentation. One or two dosages of avirulent mycobacteria at 1-2×10 6  colony forming units (CFU) have previously been used, but other dosages are contemplated within the scope of the invention. Multiple dosages can be used as needed to provide the desired level of protection from challenge. 
     It is well known in the art that in order to elicit an immune response with a live vaccine such as an avirulent mycobacteria, it is preferred that the vaccine organism can sustain an infection in the immunized host, to provide a sustained exposure of the host&#39;s immune system to the organism. Therefore, in various preferred embodiments, the  M. tuberculosis  of the invention are capable of sustaining an infection in the host. The ability to sustain infection can be measured without undue experimentation by any of a number of ways described in the art. With the mycobacteria of the present invention, a preferred way of measuring sustained infection is by determining whether viable mycobacteria of the inoculated strain will remain resident in an immunocompetent mouse (e.g., BALB/c or C57BL/6 strain) for more than four weeks. More preferably, the inoculated mycobacteria will remain resident in the mouse for at least ten weeks. In the most preferred embodiments, viable mycobacteria of the inoculated strain will remain resident in the mouse for at least 20 weeks. 
     Preferably, the attenuated mycobacteria of the invention are capable of protecting a mammal from challenge by a virulent  M. tuberculosis  complex mycobacteria. This ability can be determined by any of a number of ways provided in the literature. A preferred method is aerogenically treating an immunocompetent mouse with the virulent mycobacteria, as described in Examples 1 and 2. Aerogenic challenge is preferred because that most closely mimics natural infection. The skilled artisan would understand that the ability of an avirulent  mycobacterium  to protect a mouse from challenge from a virulent  mycobacterium  is indicative of the ability of the avirulent  mycobacterium  to protect a human, including a human child, from tuberculosis infection. A more stringent test of an avirulent  mycobacterium  to prevent infection by a virulent challenge is to use an immunocompromised mammal such as a SCID mouse. 
     The deletion of the RD1 region or the region controlling production of a vitamin is contemplated in these embodiments with any  M. tuberculosis  strain. Preferably, the strain is a virulent strain, since those strains would be most likely to sustain an infection after the deletion is made. Preferred  M. tuberculosis  strains are the H37Rv and CDC1551 strain, because the genetics of those strains are very well known. 
     In some aspects of these embodiments, the deletion is of the RD1 region (see Example 1). Strains with these deletions can be determined by any means in the art, preferably by molecular genetic means, for example by hybridization methods (e.g., Southern blot using a probe from the RD1 region) or by amplification methods (e.g., PCR using primers to amplify a portion of the RD1 region). An example of an  M. tuberculosis  RD1 region (from H37Rv) is provided herein as SEQ ID NO:1. The skilled artisan could identify analogous RD1 regions from other  M. tuberculosis  complex mycobacteria without undue experimentation. Those RD1 regions would be expected to have strong homology to SEQ ID NO:1, at least 80% homologous to SEQ ID NO:1. However, it is to be understood that virulent  M. tuberculosis  can be rendered avirulent by deletions in a portion of the RD1 region. Therefore, non-naturally occurring  M. tuberculosis  that have a partial deletion in the RD1 region are envisioned as within the scope of the invention, provided the deletion can cause a virulent  M. tuberculosis  to become avirulent. It is expected that such  M. tuberculosis  with partial RD1 deletions can still sustain an infection in a mammal and protect against challenge by a virulent  M. tuberculosis.    
     In embodiments where the deletion is in a region controlling production of a vitamin, the deletion can be in any genetic element leading to loss of production of the vitamin, including structural genes for enzymes involved in the biosynthesis of the vitamin, and genetic control elements such as promoters, enhancers, etc. 
     Deletion of a region controlling production of any essential vitamin or their precursors is contemplated as within the scope of the invention. As used herein, an essential vitamin is defined by its normal usage, that is, a small molecular weight compound that is required as a cofactor for the efficient function of an essential enzyme or enzymes. Nonlimiting examples include vitamin A, thiamin (B1), riboflavin (B2), nicotinic acid (niacin)/nicotinamide/nicotinamide adenine dinucleotide (NAD)/nicotinamide adenine dinucleotide phosphate (NADP/coenzyme II), pantothenate (pantothenic acid/B5), pyridoxine (B6), folic acid, B12, biotin, C, D, E and K. Preferred vitamin targets for deletion include nicotinamide and pantothenate (see Example 2). Methods for determining whether a  mycobacterium  has deletions leading to the loss of production of any of these vitamins are within the scope of the art. 
     Deletions leading to the loss of any of these vitamins would be expected to lead to attenuated virulence of an otherwise virulent  mycobacterium  in the  M. tuberculosis  complex. Any of those strains would also be expected to sustain an infection in a mammal. 
     Preferred vitamin targets are pantothenate and nicotinamide adenine dinucleotide (NAD) (see Example 2). A preferred pantothenate deletion is of structural genes in the pantothenate biosynthetic operon, most preferably the panC and panD genes, the combined mutation being ΔpanCD. An example of a deletion of those genes is the deletion of the sequence from  M. tuberculosis  H37Rv provided herein as SEQ ID NO:2. Similarly, a preferred NAD deletion is in the structural genes of the NAD biosynthetic operon, most preferably the nad B and C genes, the combined mutation being ΔnadBC. An example of a deletion in those genes is the deletion of the sequence from  M. tuberculosis  H37Rv provided herein as SEQ ID NO:3. 
     In similar embodiments, the invention is directed to any of the above-described  M. tuberculosis  that are produced by deleting an RD1 region or a region controlling production of a vitamin. The deletion can be made by serial in vitro passage of a virulent  M. tuberculosis  (as the well-known  M. bovis  BCG was made) and selection for the desired deletion. More preferably, however, the deletion is made by genetic engineering, since such genetic methods allow precise control of the deletion being made. 
     Various methods of making deletions in mycobacteria are known in the art. Nonlimiting examples include specialized transduction (see, e.g., U.S. Pat. No. 6,271,034, Example 1 and Example 2), and sequential two-step recombination (see Example 1). The latter method can usefully employ a sacB selective marker (Example 1). 
     Since, in preferred embodiments of the invention, the mycobacteria exhibit attenuated virulence and can sustain an infection in a mammal, these mycobacteria can usefully further employ a foreign DNA stably integrated into the genome of the mycobacteria, such that the mycobacteria can express a gene product coded by the foreign DNA. See, e.g., U.S. Pat. No. 5,504,005. 
     Thus, it is apparent that the present invention has wide applicability to the development of effective recombinant vaccines against bacterial, fungal, parasite or viral disease agents in which local immunity is important and might be a first line of defense. Non-limiting examples are recombinant vaccines for the control of bubonic plague caused by  Yersinia pestis , of gonorrhea caused by  Neisseria gonorrhoea , of syphilis caused by  Treponema pallidum , and of venereal diseases or eye infections caused by  Chlamydia trachomatis . Species of  Streptococcus  from both group A and group B, such as those species that cause sore throat or heart disease,  Neisseria meningitidis, Mycoplasma pneumoniae, Haemophilus influenzae, Bordetella pertussis, Mycobacterium leprae, Streptococcus pneumoniae, Brucella abortus, Vibrio cholerae , Shigella spp.,  Legionella pneumophila, Borrelia burgdorferi ,  Rickettsia  spp.,  Pseudomonas aeruginosa , and pathogenic  E. coli  such as ETEC, EPEC, UTEC, EHEC, and EIEC strains are additional examples of microbes within the scope of this invention from which foreign genes could be obtained for insertion into mycobacteria of the invention. Recombinant anti-viral vaccines, such as those produced against influenza viruses, are also encompassed by this invention. Recombinant anti-viral vaccines can also be produced against viruses, including RNA viruses such as Picornaviridae, Caliciviridae, Togaviridae, Flaviviridae, Coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae or Retroviridae; or DNA viruses such as Hepadnaviridae, Paroviridae, Papovaviridae, Adenoviridae, Herpesviridae or Poxyiridae. 
     Recombinant vaccines to protect against infection by pathogenic fungi, protozoa or parasites are also contemplated by this invention. 
     The avirulent microbes of the present invention are also contemplated for use to deliver and produce foreign genes that encode pharmacologically active products that might stimulate or suppress various physiological functions (i.e., growth rate, blood pressure, etc.). In such microbes, the recombinant gene encodes said pharmacologically active products. 
     By immunogenic agent is meant an agent used to stimulate the immune system of an individual, so that one or more functions of the immune system are increased and directed towards the immunogenic agent. Immunogenic agents include vaccines. 
     An antigen or immunogen is intended to mean a molecule containing one or more epitopes that can stimulate a host immune system to make a secretory, humoral and/or cellular immune response specific to that antigen. 
     In preferred embodiments, the foreign DNA encodes an antigen, an enzyme, a lymphokine, an immunopotentiator, or a reporter molecule. Preferred examples include antigens from  Mycobacterium leprae, Mycobacterium tuberculosis , malaria sporozoites, malaria merozoites, diphtheria toxoid, tetanus toxoids,  Leishmania  spp.,  Salmonella  spp.,  Mycobacterium africanum, Mycobacterium intracellulare, Mycobacterium avium ,  Treponema  spp., Pertussis, Herpes virus, Measles virus, Mumps virus,  Shigella  spp.,  Neisseria  spp.,  Borrelia  spp., rabies, polio virus, human immunodeficiency virus, snake venom, insect venom, and  Vibrio cholera ; steroid enzymes; interleukins 1 through 7; tumor necrosis factor α and β; interferon α, β, and γ; and reporter molecules luciferase, β-galactosidase, β-glucuronidase and catechol dehydrogenase. 
     The scope of the present invention includes novel mycobacteria in the  M. tuberculosis  complex that are genetically engineered to comprise a deletion of an RD1 region or a region controlling production of a vitamin. The scope of the deletions and the characteristics of these mycobacteria are as with the  M. tuberculosis  mycobacteria described above. These mycobacteria include any in the  M. tuberculosis  complex, including  M. africanum, M. bovis  including the BCG strain and the subspecies caprae,  M. canettii, M. microti, M. tuberculosis  and any other mycobacteria within the  M. tuberculosis  complex, now known or later discovered. Preferred species are  M. bovis , including the BCG strain, and  M. tuberculosis , since those species are the most important as causes of mammalian diseases, such as tuberculosis in humans and  M. bovis  infection in cows. 
     Also included as within the scope of the invention is any non-naturally occurring  mycobacterium  in the  M. tuberculosis  complex having a deletion of a region controlling production of a vitamin. These mycobacteria preferably are capable of sustaining an infection in a mammal. The scope of the deletions and the characteristics of these mycobacteria are as with the  M. tuberculosis  and other mycobacteria described above. 
     The inventors have also discovered that mycobacteria in the  M. tuberculosis  complex that are auxotrophic for lysine have attenuated virulence and protect a mammal from challenge by a virulent  mycobacterium . See Example 5. Thus, in some embodiments, the invention is directed to non-naturally occurring mycobacteria in the  M. tuberculosis  complex that comprise a deletion of a region controlling production of lysine. These mycobacteria are capable of sustaining an infection in an immunocompetent mouse for at least 20 weeks. As with previously described embodiments, these mycobacteria can be any species in the  M. tuberculosis  complex. However, due to their importance as disease organisms, it is preferred mycobacteria are  M. tuberculosis  and  M. bovis , e.g.,  M. bovis  BCG. 
     These mycobacteria would also be expected to exhibit attenuated virulence in a mammal when compared to the mycobacteria without the deletion. Additionally, they would be expected to provide protection to a mammal from challenge by a virulent  mycobacterium  in the  M. tuberculosis  complex. A preferred deletion is a ΔlysA deletion, for example as provided herein as SEQ ID NO:4. 
     When constructing a live vaccine that is an attenuated pathogen due to a deletion, it is often desirable to include a second deletion, to better assure the safety of the vaccine. Second deletions in any of the above-described mycobacteria are thus contemplated as within the scope of the invention. The second deletion preferably can also attenuate virulence of an otherwise virulent  mycobacterium  in the  M. tuberculosis  complex. This second deletion can be the RD1 region if the first deletion is not. The second deletion can also be a deletion that would cause a prototrophic  mycobacterium  to be auxotrophic, or any other deletion that could improve the safety or efficacy of the  mycobacterium  in protecting against infection. Nonlimiting examples include deletions in a gene or genes controlling production of an amino acid or a nucleotide, or a vitamin not eliminated by the first mutation. 
     The inventors have also discovered that two attenuating deletions in a  mycobacterium  in the  M. tuberculosis  complex provides a high level of protection to a mammal from challenge by a virulent  mycobacterium . See Example 6. 
     Thus, in some embodiments, the invention is directed to mycobacteria in the  M. tuberculosis  complex which are genetically engineered to comprise two deletions. Preferably, each of the two deletions are capable of individually attenuating virulence when engineered into a virulent  mycobacterium  in the  M. tuberculosis  complex. 
     Preferred embodiments of these mycobacteria are as with the other mycobacteria of the invention, e.g., the  mycobacterium  is preferably a  Mycobacterium tuberculosis ; the  mycobacterium  is preferably capable of sustaining an infection in an immunocompetent mouse for at least 20 weeks; and the  mycobacterium  is capable of protecting the mammal from challenge by a virulent  mycobacterium.    
     As with the other mycobacteria previously described, the two attenuating deletions can be any deletions that are individually capable of attenuating virulence of an otherwise virulent strain. Preferred deletions are deletions of an RD1 region (e.g., a deletion of SEQ ID NO:1), deletions of a region controlling production of a vitamin, or deletions of a region controlling the production of an amino acid, as previously discussed. A preferred deletion of a region controlling production of a vitamin is the ΔpanCD deletion, e.g., as disclosed in Examples 2 and 3, discussing attenuated strains having a deletion of SEQ ID NO:2. Preferred deletions of regions controlling production of amino acids are those regions controlling production of proline, tryptophan, leucine or lysine. See, also, Examples 5 and 6, describing strains having a ΔlysA deletion (SEQ ID NO:4), or two mutations including one with a ΔlysA deletion. 
     In additional embodiments, the invention is directed to tuberculosis vaccines made using any of the above described mycobacteria, in a pharmaceutically acceptable excipient. These vaccines are capable of protecting the mammal from challenge by a virulent  M. tuberculosis  complex mycobacteria. In some preferred embodiments, the  mycobacterium  is a  Mycobacterium bovis  and the mammal is a cow; in other preferred embodiments, the  mycobacterium  is  M. tuberculosis  and the mammal is a human, e.g., a human child. 
     By vaccine is meant an agent used to stimulate the immune system of an individual so that protection is provided against an antigen not recognized as a self-antigen by the immune system. Immunization refers to the process of inducing a continuing high level of antibody and/or cellular immune response in which T-lymphocytes can either kill the pathogen and/or activate other cells (e.g., phagocytes) to do so in an individual, which is directed against a pathogen or antigen to which the organism has been previously exposed. The phrase “immune system” refers herein to the anatomical features and mechanisms by which a mammal produces antibodies against an antigenic material which invades the cells of the individual or the extra-cellular fluid of the individual and is also intended to include cellular immune responses. In the case of antibody production, the antibody so produced can belong to any of the immunological classes, such as immunoglobulins, A, D, E, G or M. Immune responses to antigens are well studied and widely reported. A survey of immunology is provided in Elgert (1996) and Stites et al. (1991). 
     The pharmaceutical carrier or excipient in which the vaccine is suspended or dissolved may be any solvent or solid or encapsulating material. The carrier is non-toxic to the inoculated individual and compatible with the microorganism or antigenic gene product. Suitable pharmaceutical carriers are known in the art and, for example, include liquid carriers, such as normal saline and other non-toxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose. Gelatin capsules can serve as carriers for lyophilized vaccines. Adjuvants may be added to enhance the antigenicity if desired. When used for administering via the bronchial tubes, the vaccine is preferably presented in the form of an aerosol. Suitable pharmaceutical carriers and adjuvants and the preparation of dosage forms are described in, for example, Gennaro (1985). 
     Similarly, the invention is directed to methods of protecting a mammal from a virulent  mycobacterium  in the  M. tuberculosis  complex. The methods comprise treating the mammal with any of the above-described vaccines. 
     The vaccines can be administered by oral ingestion, gastric intubation, or broncho-nasal-ocular spraying, intravenous, intramuscular, intramammary, or, preferably, by subcutaneous or intradermal injection. The immunization dosages required can be determined without undue experimentation. One or two dosages of avirulent mycobacteria at 1-2×10 6  colony forming units (CFU) have previously been used, but other dosages are contemplated within the scope of the invention. Multiple dosages can be used as needed to provide the desired level of protection from challenge (see, e.g., Example 5). 
     The present invention is also directed to methods of preparing a tuberculosis vaccine. The methods comprise deleting an RD1 region or a region controlling production of a vitamin from a  mycobacterium  in the  M. tuberculosis  complex to produce any of the mycobacteria previously described. 
     Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples. 
     In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch &amp; Sambrook, “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-IV (Ausubel, R. M., ed. (1997); and “Cell Biology: A Laboratory Handbook” Volumes I-III (J. E. Celis, ed. (1994). 
     EXAMPLE 1 
       Mycobacterium Tuberculosis  Having an RD1 Deletion has Attenuated Virulence and Protects Against Tuberculosis 
     This example describes experimental methods and results that establish that deleting the RD1 region from a virulent  M. tuberculosis  attenuates the virulence of the  M. tuberculosis  in both immunocompetent and immunocompromised mice, and protects against subsequent challenge by a virulent  M. tuberculosis.    
     Materials and Methods 
     Media and Cultures. The mycobacterial strains  M. tuberculosis  H37Rv,  M. tuberculosis  Erdman and  M. bovis  BCG Pasteur were obtained from the Trudeau Culture Collection (Saranac Lake, N.Y.). They were cultured in Middlebrook 7H9 broth and 7H10 agar supplemented with 10% OADC, 0.5% glycerol, and 0.05% Tween 80. Cyclohexamide, which does not affect mycobacterial growth, was added to the 7H10 agar medium at 0.1% to avoid fungal contamination. To examine the colony morphology of mycobacteria, Tween 80 was not added to 7H10 agar medium. The acriflavin resistant strain (Hepper and Collins, 1984) of  M. tuberculosis  Erdman grew in the presence of 20 μg of acriflavin per ml of medium. 
     DNA manipulation and construction of  M tuberculosis  ΔRD1. The following four primers were used to amplify upstream and downstream flanking sequences (UFS and DFS, respectively) for the construction of the RD1 deletion mutants. UFS was amplified using TH201: GGGGGCGCACCTCAAACC (SEQ ID NO:5) and TH202: ATGTGCCAATCGTCGACCAGAA (SEQ ID NO:6). DFS was amplified using TH203: CACCCAGCCGCCCGGAT (SEQ ID NO:7), and TH204: TTCCTGATGCCGCCGTCTGA (SEQ ID NO:8). Recognition sequences for different restriction enzymes were included at the ends of each primer to enable easier manipulation. 
     The unmarked deletion mutant of  M. tuberculosis  H37Rv, mc 2 4004, was generated by transformation (Snapper et al., 1988) using a sacB counterselection (Pelocic et al., 1996; Pavelka and Jacobs, 1999). Specifically, the plasmid pJH508 was created by first cloning UFS into KpnI and XbaI sites, then cloning DFS into EcoRI and HindIII sites of pJH12, a pMV261-derived  E coli -Mycobacteria shuttle plasmid, to create pJH506 in which UFS and DFS flanked a green fluorescent protein gene (GFPuv, Clonetech) whose expression was driven by the  M. leprae  promoter 18Kd. The UFS-gfp-DFS cassette was sub-cloned into the EcoRV site of plasmid pYUB657 to create pJH508. The first homologous recombination involved the identification of hygromycin resistant colonies, resulting from the transformation of  M. tuberculosis  with pJH508. Southern analysis of the NcoI digested DNA isolated from hygromycin resistant colonies probed with UFS or DFS, confirmed the presence of a single copy of pJH508 inserted into the  M. tuberculosis  genome. The transformant identified was then grown in 7H9 broth to saturation, to allow the second homologous recombination to occur, resulting in recombinants that could be selected by plating the culture on 7H10 plates, supplemented with 3% sucrose. Both Southern analysis and PCR of the DNA isolated from sucrose resistant colonies confirmed the RD1 deletion. 
     Specialized transduction (Bardarov and Jacobs, 1999), a mycobacteriophage-based method for the delivery of homologous DNA constructs using conditionally replicating shuttle phasmids (Jacobs et al, 1987; Bardarov and Jacobs, 1999; Carriere et al., 1997), has been used successfully for  M. tuberculosis  (Glickman et al., 2000; Glickman et al., 2001; Raman et al., 2001). Specifically, a transducing phage phAEKO1 was constructed by inserting UFS and DFS into pJSC347, flanking a hygromycin cassette, to create pJH313. pJH313 was digested with PacI and ligated to phAE159, a temperature sensitive mycobacteriophage derived from TM4. The transduction was performed by growing  M. tuberculosis  to an O.D. 600  of 0.8, washing twice with MP buffer, re-suspending into an equal volume of MP buffer and mixing with the transducing phage phAEKO1 at an MOI of 10. The mixtures were incubated at 37° C. overnight, then plated on 7H10 plates supplemented with hygromycin at 50 μg/ml. Hygromycin resistant colonies were analyzed by PCR and Southern hybridization, as described above, to confirm the deletion of RD1. 
     Complemetation analyses was performed using the integration proficient cosmids (Pascopella et al., 1994; Lee et al., 1991) pYUB412 made by S. Bardarov, a library made by F. Bange, and cosmid identified and generously provided by S. T. Cole. 
     Results 
     Genetic engineering of  M. tuberculosis  mutants with RD1 deletions. The RD1 (region of difference) region has been defined as the specific 9454 bp of DNA that is present in virulent  M. tuberculosis  and  M. bovis , but absent in BCG (Mahairas et al., 1996). The annotation of RD1 predicts that the deletion would disrupt 9 genes encoding ORF&#39;s (Id.; Cole et al., 1998). Five of the 9 ORF&#39;s have no known functions (Rv3871, Rv3876, Rv3877, Rv3878 and Rv3879c), two genes encode members of the PE/PPE family (Rv3872/Rv3873), and two genes encode the secreted proteins Cfp10 (Berhet et al., 1998) and Esat6 (Andersen et al., 1991) (Rv3875) ( FIG. 1 ). To test if the RD1 region is essential for virulence in  M. tuberculosis , it was necessary to 1) delete the RD1 region from virulent  M. tuberculosis  strains, 2) demonstrate loss of virulence and 3) restore virulence by complementation with the RD1 DNA. The RD1 deletion (ΔRD1) was successfully introduced into  M. tuberculosis  by two different techniques, utilizing both a plasmid that allows two-step sequential recombination to make an unmarked deletion, and specialized transduction ( FIG. 1   a - c ). For both methods, the same 1200 bp on each side of the RD1 deletion were cloned into the appropriate plasmid or phage vector and then introduced into  M. tuberculosis  H37Rv by transformation or phage infection. An unmarked RD1 deletion mutant of  M. tuberculosis  H37Rv, mc 2 4004, was constructed, purified, and has the advantage that additional mutations can be readily added to it. In addition, the RD1 deletion was successfully engineered in the H37Rv and Erdman strains of  M. tuberculosis  using a specialized transducing phage. Since TM4 phages have been shown to infect over 500 clinical  M. tuberculosis  isolates (Jacobs et al., 1987), it should be possible to introduce the RD1 deletion into any  M. tuberculosis  isolate of interest. 
       M. tuberculosis  H37Rv ΔRD1 is attenuated for virulence. To test if the RD1 deletion causes an attenuating phenotype in  M. tuberculosis , the  M. tuberculosis  H37Rv ΔRD1 (mc 2 4004) was introduced into immunocompromised mice possessing the SCID (severe combined immunodeficiency) mutation. Groups of ten mice were injected intravenously with either 2×10 6    M. tuberculosis  H37Rv or  M. tuberculosis  H37Rv ΔRD1 and three mice per group were sacrificed 24 hours later to verify the inoculation doses. All of the SCID mice infected with the parental  M. tuberculosis  H37Rv strain died within 14 to 17 days post infection ( FIG. 2   a ). In contrast, the SCID mice infected with the same dose of  M. tuberculosis  H37Rv ΔRD1 were all alive at 35 days post-infection demonstrating a marked attenuation of the strain. To prove that the attenuation was due to the RD1 deletion, mc 2 4004 was transformed with an integrating plasmid containing the RD1 region from  M. tuberculosis  H37Rv. SCID mice injected intravenously with 2×10 6  of the transformed strain died 13 to 16 days post-infection ( FIG. 2   a ), thereby, establishing that the genes in the RD1 deletion complemented the attenuating phenotype. 
     To further characterize the attenuating phenotype of the RD1 deletion in mc 2 4004, we compared the virulence of  M. tuberculosis  H37Rv and BCG-Pasteur to  M. tuberculosis  H37Rv ΔRD1 with time-to-death experiments in SCID mice following injections with 10-fold varying inocula. Groups of 10 mice were injected intravenously, each mouse receiving from 2×10 3  to 2×10 6  CFU.  FIG. 2   b  shows that the SCID mice succumbed to the infection with all three mycobacterial strains. However, the SCID mice succumbed to an  M. tuberculosis  H37Rv intravenous infection within 2 to 5 weeks, in a dose dependent manner. In the same time frame, the SCID mice did not succumb to infection with  M. tuberculosis  H37Rv ΔRD1 until week 7, and only then, with the high dose of 2×10 6  CFU. Mice receiving 2×10 3  CFU  M. tuberculosis  H37Rv ΔRD1 survived longer than 14 weeks post infection, the survival rate of which coincided with the mice receiving 2×10 6  CFU of  M. bovis  BCG. Thus, these experiments established that  M. tuberculosis  H37Rv ΔRD1 was significantly more attenuated than its parent, but not as attenuated as BCG-Pasteur in the immunocompromised mice. 
     Colonial morphotypes of  M. tuberculosis  H37Rv ΔRD1. The  M. tuberculosis  H37Rv ΔRD1 mutant was generated independently three times from the single crossover construct (mc 2 4000) and upon subculturing, consistently yielded a 20 to 50% mixture of two colonial morphotypes on Middlebrook medium without Tween 80 ( FIG. 3   a ). One morphotype was a smooth (S) phenotype that was flat and corded (like the parental  M. tuberculosis  H37Rv strain) and the second was a rough and raised (R) phenotype. Repeated subculturing of either the R or S colonies continued to yield both colonial morphotypes, but with a distribution of approximately 80% smooth and 20% rough colonies. The distinction of these two types of morphology could be noted even when the colonies were less than two weeks old as the rough colonies were constricted and elevated with only a small portion of the base of the colony attached to the agar, while the smooth colonies tends to be flattened and spread out. When colonies grew older, e.g. 6 weeks old, the rough colonies remained more constricted compared to those of smooth colonies. The rough colonies exhibited large folds on the surface ( FIG. 3   f, g ), as compared to those of the smooth colonies that exhibited small wrinkles ( FIG. 3   e ). 
     Interestingly, in 1929, Petroff et al. reported a similar property for an early-derived BCG strain (Petroff et al., 1929) and proposed that the attenuation phenotype of BCG was not stable. Calmette disputed that the avirulent phenotype reverted and postulated that Petroff et al. had acquired a contaminating virulent strain. Southern analysis of R and S colonies revealed each morphotype has the same RD1-deleted genotype ( FIG. 3   d ). Furthermore, complementation of  M. tuberculosis  H37Rv ΔRD1 with the RD1 region restored the mutant phenotype back to the homogenous parental S phenotype (FIG.  3   a - c ). These results suggest that the variable morphotypes resulted directly from the RD1 deletion thus dissociating a direct correlation of virulence with morphotype. 
     The  M. tuberculosis  H37Rv ΔRD1 is highly attenuated in immunocompetent BALB/c mice. To further assess the pathogenicity, survival, growth kinetics, and the histopathological analysis of the  M. tuberculosis  H37Rv ΔRD1 mutant, we compared the parental  M. tuberculosis  H37Rv to BCG-Pasteur strains in BALB/c mice. In survival studies, greater than 50% BALB/c mice had died at 14 weeks post i.v. infection with 2×10 6  CFUs of  M. tuberculoisis  H37Rv strain ( FIG. 4   a ). In contrast, all mice infected with a similar dose of either BCG or  M. tuberculosis  H37Rv ΔRD1 survived for longer than 22 weeks. These results were substantiated in a separate experiment in which a group of 11 BALB/c mice were infected with 1×10 5  CFU of  M. tuberculosis  H37Rv ΔRD1 and 9 of 11 mice (81%) survived greater than 9 months post-infection (data not shown). While BCG and  M. tuberculosis  H37Rv ΔRD1 showed similar survival results, the growth relative kinetics in mouse organs differed substantially. BCG grew in a limited fashion in lungs, liver and spleen in BALB/c mice and was cleared to undetectable levels by week 12 ( FIG. 4   b - d ). In contrast, the  M. tuberculosis  H37Rv ΔRD1 strain grew in a fashion indistinguishable from the parental  M. tuberculosis  H37Rv in all mouse organs for the first 8 weeks. Thereafter, mice infected with the parental  M. tuberculosis  failed to contain the infection leading to mortality. Strikingly, mice infected with the  M. tuberculosis  H37Rv ΔRD1 showed a definite control over infection resulting in significantly prolonged survival of mice ( FIG. 4   b - d ). 
     The differing survival data of the three strains was clearly substantiated by histopathological analysis.  M. tuberculosis  H37Rv ΔRD1 caused less severe organ damage in the lung, liver and spleen than the highly virulent parent strain  M. tuberculosis  H37Rv.  M. bovis  BCG was the least virulent of the three strains. Based on histopathological evaluation, the mortality in mice infected with the wild type  M. tuberculosis  H37Rv (documented above and in  FIG. 4   a ) was caused by worsening pneumonia, hepatitis and splenitis ( FIG. 5   a - c ). Mice examined at 14 weeks post-infection had developed severe lobar granulomatous pneumonia. Acid fast staining demonstrated large numbers of  M. tuberculosis  H37Rv, often in clumps, throughout the lung. The livers and spleens showed a severe diffuse granulomatous inflammation. 
     Histopathological examination further demonstrated that  M. tuberculosis  H37Rv ΔRD1 was attenuated in virulence compared to the parent strain  M. tuberculosis  H37Rv ( FIG. 5   d - f ). In contrast to the rapidly progressive infection with the parent strain  M. tuberculosis  H37Rv, the lung lesions caused by  M. tuberculosis  H37Rv ΔRD1 were maximal in mice examined at 8 weeks post-infection. Consolidating granulomatous pneumonia involved an estimated 25-30% of the lung in these mice. Numerous organisms were demonstrated by acid fast staining. The pneumonia subsequently underwent partial resolution. By 14 weeks, and again at 22 weeks post-infection, the lungs showed peribronchial and perivascular inflammatory cell accumulations and focal, generally non-confluent, granulomas now with a prominent lymphocytic infiltration. The numbers of acid fast organisms were reduced. Liver lesions consisted of low numbers of scattered granulomas. Spleens were smaller, with persistent granulomas in the red pulp. 
     Mice infected with  M. bovis  BCG showed mild lesions in the lung, liver and spleen at all time points ( FIG. 5   g - i ). At longer time intervals post-infection the lesions were fewer in number, smaller with prominent lymphocytic infiltrations. At 14 weeks post-infection,  M. bovis  BCG was below the level of detection by acid fast staining. In summary, whereas  M. tuberculosis  H37Rv ΔRD1 initially grew in a manner similar to the parental  M. tuberculosis  H37Rv, this RD1 mutant was limited in the extent of spread of infection, particularly in the lung. This contrasted to the extensive and severe damage caused by the parent strain. The subsequent resolving granulomas, localization of the lesions and changes in the composition of the inflammatory cell infiltrations suggested that the mice mounted an effective immune response to combat  M. tuberculosis  H37Rv ΔRD1 infection and thereby reduced the numbers of viable organisms. 
       M. tuberculosis  H37Rv ΔRD1 protects mice against aerosolized  M. tuberculosis  challenge. To test the potential of  M. tuberculosis  H37Rv ΔRD1 to immunize mice and protect against tuberculous challenge, we used the model of subcutaneous immunization followed by aerosol challenge with virulent  M. tuberculosis . Our initial studies in C57BL/6 mice monitored the growth the  M. tuberculosis  H37Rv ΔRD1 strain over an 84-day period. Groups of mice (5 mice per group) were vaccinated subcutaneously (sc) either once or twice, 6 weeks apart, with 10 6  CFU  M. tuberculosis  H37Rv ΔRD1 organisms. Additional mice were infected intravenously (iv) with the same dose of the RD1-deleted strain in order to examine the pathogenicity in C57BL/6 mice. 
     As seen in Table 1,  M. tuberculosis  H37Rv ΔRD1 persisted in the lungs, liver, and spleen for 3 months at moderate levels of infection but the organisms failed to grow substantially in the lungs and spleens of mice that had been inoculated iv. In contrast, reduced persistence and decreased concentrations of  M. tuberculosis  H37Rv ΔRD1 organisms were detected in organ homogenates prepared from mice that had been vaccinated sc. For the groups of mice that had been immunized with only one dose sc., low levels of  M. tuberculosis  H37Rv ΔRD1  bacilli  were recovered from the spleen after 28 and 56 days post-vaccination; however, no splenic mycobacteria were detected 84 days after the sc. injection. Importantly, the concentration of  M. tuberculosis  H37Rv ΔRD1 organisms in the lungs after the sc. immunizations was below the threshold of detection (&lt;100 CFUs per organ) for the CFU assay at nearly all time points during the three month study. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Growth kinetics in C57BL/6 mice. 
               
            
           
           
               
               
               
            
               
                   
                 Lung (Log CFU) 
                 Spleen (Log CFU) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Weeks 
                 i.v. 
                 s.c. 
                 s.c. (2×) 
                 i.v. 
                 s.c. 
                 s.c. (2×) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 4 
                 5.86 ± 
                 &lt;2 
                 not done 
                 5.73 ± 
                 2.41 ± 
                 not done 
               
               
                   
                 0.10 
                   
                   
                 0.05 
                 0.26 
               
               
                 8 
                 5.79 ± 
                 &lt;2 
                 2.52 ± 
                 5.37 ± 
                 3.12 ± 
                 3.62 ± 
               
               
                   
                 0.07 
                   
                 0.34 
                 0.04 
                 0.40 
                 0.29 
               
               
                 12 
                 5.61 ± 
                 &lt;2 
                 &lt;2 
                 5.40 ± 
                 &lt;2 
                 3.52 ± 
               
               
                   
                 0.09 
                   
                   
                 0.05 
                   
                 0.22 
               
               
                   
               
               
                 Mice were infected with 10 6    M. tuberculosis  H73Rv ΔRD1 by different routes. 
               
               
                 The data are presented as mean ± standard error of the mean. 
               
            
           
         
       
     
     Three months after the Sc. vaccinations with the ΔRD1 strain, groups of mice were challenged aerogenically with a low dose (50 CFUs) of an acriflavin-resistant strain of  M. tuberculosis  Erdman. The use of a drug-resistant challenge strain permitted the differentiation of the challenge organisms from the sensitive vaccine population. As controls, other groups of mice were immunized Sc. with 10 6  CFUs of BCG Pasteur. The protective responses induced by the  M. tuberculosis  H37Rv ΔRD1 vaccination were evaluated by assessing the relative growth of the acriflavin-resistant challenge organisms in naive, BCG vaccinated, and  M. tuberculosis  H37Rv ΔRD1 immunized mice and by comparing the relative post-challenge lung pathology in the experimental groups and the naive controls. As seen in Table 2, the growth of the drug-resistant challenge organisms was substantially lower in the lungs of animals vaccinated with BCG or the  M. tuberculosis  H37Rv ΔRD1 vaccine. Significant reductions in the lung CFU values in the vaccinated animals (relative to naive controls) could be detected both 28 and 56 days after the challenge. Dissemination to the spleen was also significantly limited in all of the vaccination groups with the most substantial differences (−1.4 log 10  CFUs compared to the naives) being detected during the first month post-challenge. While significant differences in the growth of the mycobacterial challenge was identified between unvaccinated and vaccinated mice, the rate of proliferation of the acriflavin-resistant challenge strain in all the experimental groups (BCG sc or  M. tuberculosis  H37Rv ΔRD1 or 2 doses sc) was nearly identical and not statistically different. 
                     TABLE 2                    M. tuberculosis  ΔRD1 and BCG protect C57BL/6 mice from areosol       challenge with  M. tuberculosis  Erdman                             Lung (Log CFU)   Spleen (Log CFU)                                     Day 28   Day 56   Day 28   Day 56                                             Naive   4.77 ± 0.06   4.11 ± 0.05   3.57 ± 0.21   3.20 ± 0.16       BCG (1×)   3.96 ± 0.20   3.80 ± 0.08   2.18 ± 0.18   2.48 ± 0.23       ΔRD1 (1×)   3.97 ± 0.39   3.71 ± 0.06   2.12 ± 0.12   2.60 ± 0.25       ΔRD1 (2×)   3.96 ± 0.15   3.66 ± 0.09   2.21 ± 0.15   2.22 ± 0.16               Immunizations were performed subcutaneously once (1×) or twice (2×) with 2 × 10 6  CFUs of the vaccinating strains. Three months later, vaccinated animals were aerogenically challenged with 50 CFUs/mouse of acriflavin resistant  M. tuberculosis  Erdman. The growth of the bacterial challenge was monitored 28 and 56 days post infection by plating on Middlebrook 7H11 plates containing 20 μg/ml acriflavin and using procedures previously described (Delogu et al., 2002).            
Discussion
 
     The  M. tuberculosis  H37Rv ΔRD1 mutant strain shares significant properties with BCG including: 1) a significant attenuation of virulence in mice, 2) the ability to generate variable colonial morphotypes, and 3) the ability to protect mice against aerogenic tuberculosis challenge. These properties, and the observation that RD1 is the only deletion common to all BCG substrains, makes it likely that the RD1 deletion is the primary attenuating mutation. It remains to be determined if a single gene or a number of genes in this region causes the attenuated phenotype. The variable colonial morphotype switch does suggest that a protein regulating cell wall biogenesis is affected. Notably, defined mutations affecting the cyclopropanation of mycolic acids (Glickman et al., 2000) or the synthesis or export of phthiocerol dimycoseroate (Cox et al., 1999) have been found to correlate with decreased virulence and altered colony morphotypes in  M. tuberculosis  and thus represent attractive candidate genes that might be regulated by an RD1-encoded gene. The  M. tuberculosis  ΔRD1 mutant provides a precisely defined background strain by which to determine virulence and colony morphology related genes. 
     BCG is currently the only antituberculous vaccine available for use in humans. In many animal models, BCG has been shown to induce protective immunity against  M. tuberculosis  challenge (Opie and Freund, 1937; Hubbard et al., 1992; Baldwin et al., 1998) and in addition, has demonstrated protection against the most severe and fatal form of TB in children (Rodrigues et al., 1991). However, BCG has shown variable efficacy in protecting adults from pulmonary TB (Tuberculosis Prevention Trial, 1980; Hart and Sutherland, 1977; Bloom and Fine, 1994). Due to the uncertain efficacy of BCG, particularly in TB-endemic countries, the development of improved tuberculosis vaccines has become an international research priority. 
     Our challenge studies have demonstrated that the protective immune responses elicited by immunization with  M. tuberculosis  H37Rv ΔRD1 in mice are at least as strong as the protective responses induced by vaccination with BCG. The  M. tuberculosis  H37Rv ΔRD1 mutant also retains the BCG-associated property of limited spread to the lung following subcutaneous immunization. Restricted dissemination of the ΔRD1 mutant to the lung suggests it should have a favorable overall safety profile. Also, the unmarked mutant of  M. tuberculosis  H37Rv ΔRD1 provides a single deletion strain whereby other attenuating mutations can be readily engineered. Since the risk of reversion to wild-type virulence decreases substantially with each additional attenuating mutation,  M. tuberculosis  mutants harboring deletions in two or three separate genetic loci should provide a much safer vaccine for long term use. 
       M. tuberculosis  mutants with RD1 deletions represent attractive candidates as novel vaccines for TB prevention. These mutants, derived from a single mutagenic event from the parental  M. tuberculosis  strain, replicate more efficiently in vivo than BCG, especially early in infection. This enhanced rate of proliferation for the RD1-deleted strains, relative to BCG, may lead to the induction of increased protective immunity in humans, after vaccination with  M. tuberculosis  H37Rv ΔRD1. Moreover, they could also be more immunogenic as there exist at least 129 ORFs present in  M. tuberculosis  H37Rv that are absent from  M. bovis  (Behr et al., 1999). Since some of these ORFs are likely to encode regulatory proteins affecting the expression of other genes, there could be hundreds of antigens expressed in  M. tuberculosis -infected cells that are absent from BCG-infected cells. Thus, RD1 deletion mutants constructed from human  tubercle bacilli  could protect humans against disease substantially better than BCG. 
     EXAMPLE 2 
     Vitamin Auxotrophs of  Mycobacterium Tuberculosis  are Attenuated and Protect Against Tuberculosis 
     This example describes experimental methods and results that establish that deleting genes that control vitamin production in a virulent  M. tuberculosis  causes the  M. tuberculosis  to become avirulent and sustain an infection in mammals, and protect the mammal against challenge with a virulent  M. tuberculosis.    
     Given the importance of NAD and nicotinamide (vitamin B3) and pantothenate (vitamin B5) as cofactors involved in carbon utilization, energy transduction (Abiko, 1975; Jackowski, 1996) and the biosynthesis of the complex lipid cell wall of  M. tuberculosis , we hypothesized that mutations in the biosynthetic pathways for NAD and pantothenate could lead to the generation of mutant strains that retain a limited ability to replicate and subsequently get cleared within the host tissues. In  M. tuberculosis , the nadABC operon controls the de novo biosynthesis of NAD. Similarly, the panC and panD genes that are organized in an operon control the rate-limiting step in the de novo biosynthesis of pantothenate. We constructed deletion mutants of  M. tuberculosis  in the nadBC and panCD genes using specialized transduction, as described in Example 1. The mutant strains mc 2 3122 (ΔnadBC) and mc 2 6001 (ΔpanCD) were auxotrophic for nicotinamide and pantothenate respectively. The in vitro reversion frequencies of the respective mutations were found to be less than 10 −10  events per generation. 
     The safety and attenuation of ΔnadBC and ΔpanCD auxotrophic mutants were assessed by infection of immune-compromised SCID mice. SCID mice infected with wild-type  M. tuberculosis  and the ΔnadBC mutant succumbed to infection in about 5 weeks (data not shown). This result clearly indicates that in the absence of T-cell immunity, intermediates of NAD biosynthetic pathway, such as nicotinamide, are readily available in the macrophages to support the growth of the ΔnadBC mutant. In contrast all mice infected with the ΔpanCD mutant survived longer than 30 weeks, demonstrating the severe attenuation of this mutant strain. The full virulence phenotype was restored when the panCD wild type alleles were integrated into the chromosome of the ΔpanCD mutant in single copy, suggesting the observed attenuation in ΔpanCD to be due to the requirement of pantothenate for growth and not due to polar effects of the mutation on downstream genes. SCID mice infected with the same dose of conventional BCG-Pasteur vaccine strain succumbed to infection within 80 days ( FIG. 6A ) in accordance with earlier reports (Guleria, 1996). Enumeration of bacterial burdens in SCID mice infected with wild type  M. tuberculosis  H37Rv and the complementing strain (panCD in single copy integrated into the chromosome) showed a rapid increase in bacterial numbers in spleen, liver and lung before they succumbed to infection. In contrast, mice infected with ΔpanCD mutant, showed an initial drop in bacterial numbers in spleen and liver followed by a steady increase to reach 10 8  in the lungs at 160 days, at which time all mice were still alive ( FIG. 6B ). 
     Having demonstrated the significant attenuation of ΔpanCD mutant, we sought to address the in vivo growth characteristics of this mutant in immune-competent BALB/c mice. All BALB/c mice infected with H37Rv succumbed to infection by day 25 with a MST of 22 days. Similarly, mice infected with the panCD-complemented strain were highly virulent with 100% mortality between 3-8 weeks post-infection similar to the wild type strain, with a MST of 28 days. In contrast, all mice infected with ΔpanCD mutant survived for over 33 weeks demonstrating the severe attenuation phenotype of this mutant in immune-competent mice ( FIG. 7A ). Interestingly, bacterial enumeration at three weeks post infection showed 1 log increase in the ΔpanCD numbers in lungs followed by a state of persistence with the onset of adaptive immune response. This growth characteristic was observed only in the lung but not in spleen or liver (FIG.  7 B,C). A desirable trait of an effective live attenuated vaccine strain is its ability to grow within the host in a limited fashion in order to produce in vivo all the important protective antigens (McKenney, 1999; McKenny, 2000; Kanai, 1955). The ΔpanCD mutant exhibits this characteristic in the lung, which is the primary site of infection in humans and does not get cleared over a prolonged period in all the three organs. The earlier auxotrophs of  M. tuberculosis  failed to grow in any of the organs and hence failed to adequately protect against experimental challenge in guinea pigs (Jackson, 1999), or mice. 
     The ability of the ΔpanCD mutant to exhibit limited growth in the lung until the onset of adaptive immune response suggests that an unidentified putative pantothenate permease is able to transport this nutrient into resting macrophages, as in the SCID mice. A sodfum-dependent pantothenate permease actively transports pantothenate into the cell of  Escherichia coli  (Vallari and Rock, 1985; Jackowski and Alix, 1990),  Plasmodium falciparum  (Saliba and Kirk, 2001) and mammals. Subsequent activation of macrophages leads to restricted supply of this nutrient within the phagosome resulting in growth arrest of the mutant. Pantothenic acid or its derivatives have been reported to confer resistance to radiation and oxidative stress by virtue of their role in biosynthesis of CoA and also by indirectly increasing the cellular supply of glutamate, a precursor of glutathione (Slyshenkov, 1995). Pantothenate kinase (PanK) mutants of Drosophila display membrane defects and improper mitosis and meiosis due to decreased phospholipid biosynthesis (Afshar et al., 2001). The disruption of de novo pantothenate biosynthesis causes an increased susceptibility of the ΔpanCD mutant to reactive oxygen and nitrogen intermediates that are released within activated macrophages. 
     Having observed the ΔnadBC mutant to be non-attenuated in SCID mice, we chose to study the in vivo growth kinetics of this mutant in the more resistant C57BL/6 mice background. During the first three weeks of infection, the number of wild type and mutant bacteria recovered from all three organs showed little or no difference. Their numbers gradually increased in the lungs to reach 10 6 . However, with the onset of adaptive immune response at three weeks, when the growth of bacteria in the lungs of mice infected with H37Rv became constant and tightly controlled, bacterial load in the lungs of mice infected with ΔnadBC mutant showed a constant tendency for clearance to reach more than 1.5 log drop in the bacterial numbers compared to mice infected with wild type strain ( FIG. 8A ). This difference was preserved up to 24 weeks following infection. 
     The reduced ability of the ΔnadBC mutant to sustain an infection was accompanied by attenuated virulence clearly seen from the survival experiment ( FIG. 8C ). While all mice infected with the wild type strain succumbed to infection between day 90 and 179 (MST 116 days) all mice infected with the ΔnadBC mutant (n=12) remain alive for a period of more than 8 months ( FIG. 8C ). 
     Our observation of the attenuation phenotype of ΔnadBC mutant became obvious only after the onset of immune response, suggesting that once the macrophages become activated, they restrict the amount of available NAD or NAD intermediates causing a restricted growth of the mutant strain. This would be in agreement with the recently reported observations that a significant part of antimicrobial function of the macrophages could be attributed to the IFN-γ promoted enhanced expression of indolamine 2-oxygenase (IDO), the inducible enzyme controlling L-tryptophan catabolic pathway causing an almost complete depletion of L-tryptophan pool. The enhanced catabolism of L-tryptophan leads to increased de novo biosynthesis of NAD needed to protect the cells from the free radicals formed as a result of macrophage activation. Recently, several studies have demonstrated the involvement of the tryptophan catabolism in the antimicrobial mechanisms of the activated macrophages. Induction of IDO was found responsible for the inhibition of intracellular growth of  Toxoplasma, Leishmania, Legionella  and  Chlamydia . The restricted intracellular growth of ΔnadBC mutant could be explained with the very little amount of free NAD or NAD intermediates available within the activated macrophages. 
     Having established the safety and persistence of ΔpanCD and ΔnadBC in immunocompetent mice, the protective efficacy of these mutants were evaluated using an aerosol challenge model with virulent  M. tuberculosis , using the methods described in Example 1. The aerosol route of infection was chosen, as this is the natural route of infection in humans. To assess the capacity of the auxotrophic vaccines to restrict growth of virulent  M. tuberculosis  in the organs of infected mice, bacterial numbers were enumerated one month post-infection in lung and spleen. See Table 3. In the unimmunized controls, bacterial numbers rose rapidly in the spleen and lungs, in contrast mice infected with a single dose of ΔpanCD displayed significant reduction in bacterial numbers in the spleen and lung (p&lt;0.05, in comparison to unimmunized controls). Mice given two doses of ΔpanCD displayed a statistically significant reduction in the bacterial numbers to 4.5 log units in the lung (p&lt;0.01) and 3.7 log units in the spleen (p&lt;0.05). Mice vaccinated with BCG showed comparable reduction in bacterial burden in the lung and spleen to 3.3 log units and 4.7 log units respectively (p&lt;0.01). Mice immunized with one or two doses of ΔnadBC mutant conferred statistically significant protection (p&lt;0.01 in comparison to unimmunized group) that is comparable to the protection afforded by BCG vaccination. Interestingly, mice immunized with the ΔnadBC mutant showed no detectable CFUs in the spleen suggesting that the vaccination completely prevented the hematogenous spread of wild type  M. tuberculosis  following aerosol challenge. 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Experimental Group 
                 Lung CFUs (log 10 ) 
                 Spleen CFUs (log 10 ) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 A 
               
            
           
           
               
               
               
            
               
                 Naive 
                 4.05 ± 0.21 
                 3.94 ± 0.21 
               
               
                 ΔnadBC (1 × sc) 
                 3.37 ± 0.40** 
                 &lt;2** 
               
               
                 ΔnadBC (2 × sc) 
                  3.6 ± 0.35** 
                 &lt;2** 
               
               
                 BCG (1 × sc) 
                 3.46 ± 0.19** 
                 &lt;2** 
               
            
           
           
               
            
               
                 B 
               
            
           
           
               
               
               
            
               
                 Naive 
                 5.56 ± 0.05 
                 4.35 ± 0.21 
               
               
                 ΔpanCD (1 × sc) 
                 4.99 ± 0.17 (−0.57)* 
                 3.65 ± 0.15 (−0.70)* 
               
               
                 ΔpanCD (2 × sc) 
                 4.55 ± 0.09 (−1.01)** 
                 3.73 ± 0.21 (−0.62)* 
               
               
                 BCG (1 × sc) 
                 4.71 ± 0.21 (−0.85)** 
                 3.35 ± 0.20 (−1.00)** 
               
               
                   
               
               
                 p &lt; 0.05 compared to naïve, 
               
               
                 **p &lt; 0.01 compared to naïve 
               
            
           
         
       
     
     Table 3. The attenuated  M. tuberculosis  ΔnadBC and ΔpanCD mutants protect against aerogenic challenge with  M. tuberculosis  Erdman. Groups of C57BL/6 mice (5 mice per group) were vaccinated subcutaneously either once or twice (6 weeks apart) with 10 6  CFUs of mutant strains. Control mice were vaccinated subcutaneously with 10 6  CFUs of BCG-Pasteur. Three months after the initial immunization with either ΔnadBC or ΔpanCD mutant or BCG, the mice were aerogenically challenged with approximatedly 100 CFUs of acriflavin-resistant  M. tuberculosis  Erdman (Ac r MTB) strain as described earlier (Collins, 1985) After 28 days, the challenged mice were sacrificed, and the lungs and spleens of individual mice were removed aseptically and homogenized separately in 5 ml of Tween 80-saline using a Seward stomacher 80 blender (Tekmar, Cincinnati, Ohio). The homogenates were diluted serially in Tween 80 saline and plated on Middlebrook 7H11 agar with or without appropriate supplements as required. Samples from the BCG-vaccinated controls were plated on 7H11 agar containing 2 mg of thiophenecarboxylic acid hydrazide (Sigma Chemical Co., St Louis, Mo.) per ml to inhibit growth of any residual BCG. The CFU results were evaluated using the one-way ANOVA analysis of the Graph Pad InStat program. The numbers in paranthesis represent the differences between naïve and vaccinated organ CFUs. 
     In order to test the ability of the auxotrophic mutants to confer long lasting immunity, mice were challenged 7 months after an initial subcutaneous immunization with the ΔnadBC mutant. See Table 4. Mice immunized with ΔnadBC displayed significantly reduced numbers of the challenge organism in the lungs and no detectable numbers in the spleen comparable to the numbers seen in the BCG vaccinated mice. The results suggest that the ΔnadBC vaccine strain is able to persist within the mouse organs sufficiently long to mount a long lasting immunity to control subsequent infection. 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Experimental Group 
                 Lung CFUs (log 10 ) 
                 Spleen CFUs (log 10 ) 
               
               
                   
               
             
            
               
                 Naive 
                 4.61 ± 0.07 
                 4.07 ± 0.20 
               
               
                 BCG 
                 4.00 ± 0.13* 
                  2 
               
               
                 NAD (1 × iv) 
                 3.28 ± 0.15** 
                 &lt;2 
               
               
                 NAD (2 × iv) 
                 2.95 ± 0.14** 
                 &lt;2 
               
               
                 NAD (1 × sc) 
                 4.05 ± 0.12* 
                 &lt;2 
               
               
                 NAD (2 × sc) 
                 3.94 ± 0.13* 
                 &lt;2 
               
               
                   
               
               
                 *P &lt; 0.05; 
               
               
                 **P &lt; 0.01 by Dunnett&#39;s Multiple Comparison Test 
               
            
           
         
       
     
     Table 4. Immunizations with the ΔnadBC mutant confer long-term protection against an aerosol challenge. Groups of C57BL/6 mice (5 mice per group) were vaccinated subcutaneously or intravenously either once or twice (6 weeks apart) with 10 6  CFUs of ΔnadBC mutant. Control mice were vaccinated subcutaneously with 10 6  CFUs of BCG-Pasteur. Seven months after the initial immunization with either ΔnadBC mutant or BCG, the mice were aerogenically challenged with approximately 50 CFUs of acriflavin-resistant  M. tuberculosis  Erdman (Ac r MTB) strain and the bacterial numbers at 28 days post challenge enumerated as described in Table 1. 
     To the best of our knowledge this is the first report of any  M. tuberculosis  auxotrophic vaccines administered subcutaneously to confer protection comparable to the conventional BCG vaccine strain in a mouse model of infection. Mice vaccinated with the ΔpanCD and ΔnadBC survived for over one year following the aerosol challenge indicating the protection and safety of these vaccine strains. 
     EXAMPLE 3 
     A Pantothenate Auxotroph of  Mycobacterium Tuberculosis  is Highly Attenuated and Protects Mice Against Tuberculosis 
     This Example is published as Sambandamurthy et al., 2002. Example summary. 
     With the advent of HIV and the widespread emergence of drug resistant strains of  Mycobacterium tuberculosis , newer control strategies in the form of a better vaccine could decrease the global incidence of tuberculosis. A desirable trait in an effective live attenuated vaccine strain is its ability to persist within the host in a limited fashion in order to produce important protective antigens in vivo (Kanai and Yanagisawa, 1955; McKenney et al., 1999). Rationally attenuated  M. tuberculosis  vaccine candidates have been constructed by deleting genes required for growth in mice (Jackson et al., 1999; Hondalus et al., 2000; Smith et al., 2001). These candidate vaccines failed to elicit adequate protective immunity in animal models, due to their inability to persist sufficiently long within the host tissues. Here we report that an auxotrophic mutant of  M. tuberculosis  defective in the de novo biosynthesis of pantothenic acid (vitamin B5) is highly attenuated in immunocompromised SCID mice and in immunocompetent BALB/c mice. SCID mice infected with the pantothenate auxotroph survived significantly longer than mice infected with either BCG vaccine or virulent  M. tuberculosis  (250 days, vs. 77 days, vs. 35 days). Subcutaneous immunization with this auxotroph conferred protection in C57BL/6J mice against an aerosol challenge with virulent  M. tuberculosis , which was comparable to that afforded by BCG vaccination. Our findings highlight the importance of de novo pantothenate biosynthesis in limiting the intracellular survival and pathogenesis of  M. tuberculosis  without reducing its immunogenicity in vaccinated mice. 
     Materials and Methods 
     Media and Strains.  M. tuberculosis  H37Rv,  M. tuberculosis  Erdman and  M. bovis  BCG Pasteur were obtained from the Trudeau Culture Collection (Saranac Lake, N.Y.) and cultured in Middlebrook 7H9 broth and 7H11 agar supplemented with 10% OADC, 0.5% glycerol, and 0.05% Tween 80. When required, pantothenate (24 μg/ml), hygromycin (50 μg/ml) or kanamycin (25 μg/ml) was added. Stock strains were grown in Middlebrook 7H9 broth in roller bottles and harvested in mid-logarithmic growth phase, before being stored in 1 ml vials at −70° C. until required. 
     Disruption of panCD genes in  M. tuberculosis . Specialized transduction was employed to disrupt the chromosomal copy of the panCD genes as described (U.S. Pat. No. 6,271,034). Briefly, the 823 bp region upstream to the panC gene was amplified using primers Pan1 (5′-GTGCAGCGCCATCTCTCA-3′)(SEQ ID NO:9)and Pan2 (5′-GTTCACCGGGATGGAACG-3′)(SEQ ID NO:10). A 716 bp region downstream to the panD gene was amplified using primers Pan3 (5′-CCCGGCTCGGTGTGGGAT-3′) (SEQ ID NO:11) and Pan4 (5′-GCGCGGTATGCCCGGTAG-3′)(SEQ ID NO:12). PCR products were cloned with the TOPO TA cloning kit (Invitrogen, Calif.), and sequenced. PCR products were subsequently cloned into pJSC347, flanking a hygromycin cassette to create pSKV1. PacI digested pSKV1 was ligated into the temperature-sensitive mycobacteriophage phAE159 derived from TM4 and transduced as described earlier (Glickman et al., 2000; Raman et al., 2001). Genomic DNAs from hygromycin-resistant and pantothenate-requiring colonies were digested with BssHII, and probed with a 716 bp downstream region, flanking the  M. tuberculosis  panCD operon to confirm the deletion. For complementation, the  M. tuberculosis  panCD operon was amplified by PCR from genomic DNA with its putative promoter, cloned with TA cloning kit, sequenced, and subcloned into pMV306kan, a site-specific integrating mycobacterial vector. 
     Animal infections. C57BL/6, BALB/cJ and BALB/c SCID mice (6-8 weeks old) were purchased from Jackson Laboratories and were infected intravenously through the lateral tail vein. For time-to-death assays, BALB/c SCID mice were infected intravenously with 1×10 2  CFU of  M. tuberculosis  H37Rv, 1×10 2  CFU of panCD-complemented strain, 1×10 5  CFU of ΔpanCD mutant, or 1×10 5  CFU of  M. bovis  BCG-P. For mouse organ CFU assays, BALB/cJ mice were infected with 1×10 6  CFU of  M. tuberculosis  H37Rv or the panCD-complemented strain or the ΔpanCD mutant. At appropriate time points, groups of 4-5 mice were sacrificed and the selected organs were homogenized separately in PBS/0.05% Tween 80, and colonies were enumerated on 7H11 plates grown at 37° C. for 3-4 weeks (see McKinney et al., 2000). Pathological examination was performed on tissues fixed in 10% buffered formalin. The CFU results were evaluated using the one-way ANOVA analysis of the Graph Pad InStat program. All animals were maintained in accordance with protocols approved by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee. 
     Vaccination Studies. Groups of C57BL/6 mice (5 mice per group) were vaccinated subcutaneously either once or twice (6 weeks apart) with 1×10 6  CFU of the ΔpanCD mutant strain. Control mice were vaccinated subcutaneously with 1×10 6  CFU of  M. bovis  BCG-P. Three months after the initial immunization with either the ΔpanCD mutant or BCG, the mice were aerogenically challenged with approximately 50-100 CFU of  M. tuberculosis  Erdman strain as described earlier. At 28 days following aerosol challenge, the challenged mice were sacrificed; the lungs and spleens of individual mice were removed aseptically and homogenized separately in 5 ml of Tween 80-saline using a Seward Stomacher 80 blender (Tekmar, Cincinnati, Ohio). The homogenates were diluted serially in Tween 80 saline and plated on Middlebrook 7H11 agar with or without appropriate supplements as required. Samples from the BCG-vaccinated controls were plated on 7H11 agar containing 2 mg/ml of thiophene-2-carboxylic acid hydrazide (Sigma) to inhibit growth of any residual BCG. 
     Results and Discussion 
     Lipid biosynthesis and metabolism have been shown to play a pivotal role in the intracellular replication and persistence of  M. tuberculosis  (Cox et al., 1999; Camacho et al., 1999; Glickman et al., 2000; De Voss et al., 2000; Manca et al., 2001; McKinney et al., 2000). Therefore, we sought to globally impair the ability of this bacterium to synthesize lipids. Pantothenic acid (vitamin B5) is an essential molecule required for the synthesis of coenzyme A (CoA) and acyl carrier protein (ACP), that play important roles as acyl group carriers in fatty acid metabolism, the tricarboxylic acid cycle, biosynthesis of polyketides and several other reactions associated with intermediary metabolism (Jackowski, 1996). Bacteria, plants and fungi synthesize pantothenate from amino acid intermediates, whereas it is a nutritional requirement in higher animals ( FIG. 9   a ). 
     We constructed a double deletion mutant of  M. tuberculosis  in the panC and panD genes that are involved in the de novo biosynthesis of pantothenate ( FIG. 9   b,c ). The ΔpanCD mutant was found to be auxotrophic for pantothenate with no detectable reversion to prototrophy when 1×10 10  cells were plated on minimal medium. The growth rate of the mutant was identical to wild type H37Rv in broth cultures in the presence of exogenous pantothenate (data not shown). The attenuation of the ΔpanCD mutant was assessed by infection of immunocompromised SCID mice. SCID mice infected intravenously with H37Rv succumbed to the resulting infection in about 5 weeks. In contrast, all mice infected with the ΔpanCD mutant survived for more than 36 weeks (average, 253 days) ( FIG. 10   a ). This attenuation is due to pantothenate auxotrophy as the full virulence phenotype was restored when the panCD wild type genes were integrated into the chromosome of the ΔpanCD mutant in single copy. Enumeration of bacterial burdens in SCID mice infected with H37Rv and the ΔpanCD-complemented strain showed a rapid increase in bacterial numbers in the spleen, liver and lung, until they succumbed to infection. In contrast, mice infected with the ΔpanCD mutant showed an initial drop in bacterial numbers in the spleen and liver followed by a gradual increase in the number of viable bacteria, reaching 1×10 6  colony-forming units (CFU) by day 224 ( FIG. 10   b ). Notably, the CFU values increased to 1×10 8  in the lungs of the infected mice. The ability of ΔpanCD-infected SCID mice to survive despite a substantial bacterial burden in their lungs emphasizes the extent of attenuation in this mutant and compares with the phenotype observed with the  M. tuberculosis  whiB3 and sigH mutants described recently (Steyn et al., 2000; Kaushal, 2000). Notably, SCID mice infected with bacille Calmette-Guerin-Pasteur (BCG-P) strain succumbed to infection by 83 days (Weber et al., 2000) in contrast to the prolonged survival observed in ΔpanCD-infected mice. 
     Studies in immunocompetent mice further demonstrate the attenuation of the ΔpanCD mutant. Survival studies showed that BALB/c mice infected with H37Rv succumbed to infection by day 25 (average, 21 days) and mice infected with an identical dose of the panCD-complemented strain succumbed to infection between days 21 to 53 (average, 37 days). Importantly, all mice infected with 1×10 6  CFU of the ΔpanCD mutant survived 375 days, when the experiment was terminated ( FIG. 10   c ). At 3 weeks post infection, in contrast to the H37Rv strain, BALB/c mice infected with ΔpanCD mutant showed a 10-fold increase in bacterial numbers in the lungs followed by a gradual decline in viable numbers over the next 38 weeks of infection ( FIG. 10   d ) and the bacterial burden gradually declined in the spleen and liver throughout the course of infection ( FIG. 10   e ). Histopathologic examination of the lungs from mice infected with either H37Rv or the ΔpanCb-complemented strain, showed severe, diffuse lobar granulomatous pneumonia ( FIGS. 11   a,b ). The pneumonia affected more than 50% of the lung, and was pyogranulomatous with marked necrosis in the advanced consolidated areas, particularly in the lungs of mice challenged with H37Rv. Both of these strains caused severe granulomatous splenitis and widespread granulomatous hepatitis. At 3 weeks post-infection with the ΔpanCD mutant, low to moderate numbers of focal infiltrates of mononuclear cells scattered throughout the lung were seen ( FIG. 11   c ). The spleen was moderately enlarged with scattered granulomas. Similarly, the liver showed numerous focal granulomas. At 24 weeks post-infection, consistent with the bacterial numbers, histological examination of the lungs from mice infected with the ΔpanCD mutant showed only occasional focal mild infiltrations, predominately lymphocytic ( FIG. 11   d ). The spleen showed only mild histiocytic hyperplasia and there were fewer, focal, predominately lymphocytic accumulations in the liver. 
     The mechanisms that allow the persistence of the ΔpanCD mutant bacteria for over 8 months in the SCID mouse model remain unclear. We speculate the functional role of an unidentified permease in transporting adequate amount of pantothenate in the ΔpanCD mutant that allows its persistence but not the ability to cause disease. A pantothenate permease that transports pantothenate have been described in  Plasmodium falciparum  and  Escherichia coli  (Saliba and Kirk, 2001; Jackowski and Alix, 1990). In the lungs of immunocompetent mice, an initial growth of the ΔpanCD mutant during the first 3 weeks is followed by a steady decline in bacterial numbers following the onset of an adaptive immune response. The intracellular lifestyle of  M. tuberculosis  poses significant challenges to the bacterium in acquiring essential nutrients. Pantothenic acid or its derivatives have been shown to confer resistance to oxidative stress (Slyshenkov et al., 1996) and lack of pantothenate biosynthesis in the ΔpanCD mutant may render it more susceptible to such adverse effects. Likewise, a pantothenate kinase (pank) mutant of Drosophila was shown to display membrane defects and improper mitosis and meiosis due to decreased phospholipid biosynthesis (Afshar et al., 2001). Therefore, it is plausible that the pantothenate salvage pathway is inadequate in restoring full virulence of the ΔpanCD mutant in the absence of a functional de novo biosynthetic pathway. 
     As a test of vaccine potential, immunized mice were challenged with virulent  M. tuberculosis  Erdman by the aerosol route (Collins, 1985). Following subcutaneous immunization, the ΔpanCD mutant could not be detected in the spleens or lungs of mice at 8 and 12 weeks. In the naive controls, the bacterial CFU values increased 10,000-fold in the lung during the first month after challenge. Similarly, substantial dissemination and growth in the spleen was detected within one month of the challenge in naive controls. In contrast, mice immunized with single or double doses of the ΔpanCD mutant displayed statistically significant reductions (P&lt;0.05) in lung and spleen CFU values relative to naive controls. Mice vaccinated with BCG showed similar reduction in organ bacterial burdens compared to the nonimmunized controls ( FIG. 11   e,f ). In these aerogenic challenge studies, no significant differences were detected in the lung and spleen CFU values for mice vaccinated with either the ΔpanCD mutant strain or with BCG. At 28 days after the aerogenic challenge with virulent  M. tuberculosis , histopathological examination of lungs of ΔpanCD immunized mice revealed a less severe infection relative to the unvaccinated control mice. In controls, severe bronchitis, moderate pneumonia, and spread of the infection to the adjacent lung parenchyma was observed. By comparison, the ΔpanCD vaccinated mice had milder bronchitis and smaller areas of mild interstitial pneumonitis, with localized areas of granulomatous pneumonia in some mice. Importantly, no lung pathology was detected in vaccinated mice at the time of challenge (data not shown). Two groups of mice that were vaccinated with one or two doses of the ΔpanCD mutant and then challenged with  M. tuberculosis  Erdman were active and healthy for more than one year following the virulent challenge. Histopathological analysis of lung sections from these mice showed only mild inflammation and fibrosis despite the chronic infection. 
     By creating a  M. tuberculosis  strain that is defective in pantothenate biosynthesis, we have taken a critical step in the rational development of an attenuated  M. tuberculosis  vaccine strain. We have shown that a functional pantothenate biosynthetic pathway, which is required for the synthesis of complex mycobacterial lipids, is essential for the virulence of  M. tuberculosis . Although the precise mechanism of the reduced virulence is unclear, it is reasonable to speculate that this could be due to reduced synthesis of toxic polyketides and secreted lipids or a general slow down of metabolism.  Tubercle bacilli  lacking the two genes required to synthesize pantothenate failed to revert and were highly attenuated and less virulent than BCG vaccine when tested in the rigorous SCID mouse model of infection. Despite the reduced virulence associated with the deletion of the panCD genes, these vitamin auxotrophs remain persistent in vivo as shown by their ability to survive for at least eight months in immunocompetent mice. The persistence of this mutant strain undoubtedly contributes to the substantial immunogenicity seen in the mouse tuberculous challenge model. Overall, the ΔpanCD mutant has many of the characteristics necessary for a live vaccine candidate strain: it is attenuated by a non-reverting mutation and essentially avirulent while being persistent and immunogenic. Given the genetic differences between  M. bovis  and  M. tuberculosis  (Behr et al., 1999), one would predict that a rationally attenuated  M. tuberculosis  strain would have a more relevant repertoire of species-specific antigens and thus should elicit, in humans, more effective protective immune responses against tuberculous infections than BCG. 
     EXAMPLE 4 
     The Primary Mechanism of Attenuation of BCG is a Loss of Invasiveness Due to Host Cell Lysis 
     Example Summary 
     Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. BCG, the tuberculosis vaccine, is an attenuated mutant of  M. bovis  that was isolated following serial subcultivations, yet the basis for this attenuation has never been elucidated. A single region (RD1), deleted in all BCG substrains, was deleted from virulent  M. bovis  and  M. tuberculosis  strains and the resulting three ΔRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. Like BCG,  M. tuberculosis  ΔRD1 mutants protect mice against acrosolized  M. tuberculosis  challenge and these mutants also consistently display altered colonial morphotypes. Interestingly, the ΔRD1 mutants failed to cause necrosis, via lysis, of pneumocytes, a phenotype that had been previously used to distinguish virulent  M. tuberculosis  from BCG. We conclude that the primary attenuating mechanism of BCG is the loss of cytolytic activity, resulting in reduced invasiveness. 
     Introduction 
     BCG (bacille Calmette and Guerin), was first isolated from  M. bovis  following serial subculturing of  M. bovis  in 1908 (Calmette and Guerin, 1909). Drs. Calmette and Guerin set out to test the hypothesis that a bovine tubercle  bacillus  could transmit pulmonary tuberculosis following oral administration (Calmette and Guerin, 1905; Gheorghiu, 1996) and developed a medium containing beef bile that enabled the preparation of fine homogenous bacillary suspensions. After the 39th passage, the strain was found to be unable to kill experimental animals (Calmette and Guerin, 1909). Between 1908 and 1921, the strain showed no reversion to virulence after 230 passages on bile potato medium (Gheorghiu, 1996), which is consistent with the attenuating mutation being a deletion mutation. In the animal studies that followed, BCG was shown to be attenuated, but it also protected animals receiving a lethal challenge of virulent  tubercle bacilli  (Calmette and Guerin, 1920). BCG was first used as a vaccine against tuberculosis in a child in 1921 (Weill-Halle and Turpin, 1925). From 1921 to 1927, BCG was shown to have protective efficacy against TB in a study on children (Id.; Calmette and Plotz, 1929) and was adopted by the League of Nations in 1928 for widespread use in the prevention of tuberculosis. By the 1950&#39;s, after a series of clinical trials, the WHO was encouraging widespread use of BCG vaccine throughout the world (Fine and Rodrigues, 1990). Although an estimated 3 billion doses have been used to vaccinate the human population against tuberculosis; the mechanism that causes BCG&#39;s attenuation remains unknown. 
     Mahairas et al.(1996) first compared the genomic sequences of BCG and  M. bovis  using subtractive hybridization and found that there were three Regions of Difference (designated RD1, RD2, and RD3) present in the genome of  M. bovis , but missing in BCG. Behr et al. (Behr et al., 1999) and others (Gordon et al., 2001) later identified 16 large deletions, including RD1 to RD3, present in the BCG genome but absent in  M. tuberculosis . Eleven of these 16 deletions were unique to  M. bovis , while the remaining 5 deletions were unique to BCG. One of these 5 deletions, designated RD1 (9454 bp), was absent from all of the BCG substrains currently used as TB vaccines worldwide and it was concluded that the deletion of RD1 appeared to have occurred very early during the development of BCG, probably prior to 1921 (Behr et al., 1999). It is reasonable to hypothesize that RD1 was the primary attenuating mutation first isolated by Calmette and Guerin to generate BCG from  M. bovis . Attempts to restore virulence to BCG with RD1-complementing clones have been unsuccessful (Mahairas et al., 1996). 
     Results 
     RD1 deletions of  M. bovis  and  M. tuberculosis  are attenuated for virulence in immunocompromised mice. To test if RD1 is essential for virulence in  M. bovis  and  M. tuberculosis , it was necessary to delete the RD1 ( FIG. 1   a ) from virulent strains, demonstrate loss of virulence, and then restore virulence by complementation with the RD1 DNA. Since the original  M. bovis  parent of BCG was lost in World War I (Grange et al., 1983), we initiated studies with virulent  M. bovis  Ravenel and a variety of virulent  M. tuberculosis  strains. Despite success in generating an unmarked deletion mutant of RD1 in  M. tuberculosis  with a plasmid transformation system 1,2 , over 100 independent transformations failed to yield an RD1 deletion in  M. bovis . As an alternative strategy, specialized transduction (Bardarov et al., 2002) 3  was successfully used to generate RD1 deletion mutants not only in  M. bovis  Ravenel, but also the H37Rv, Erdman, and CDC1551 strains of  M. tuberculosis  ( FIG. 12 ). This deletion represents the largest deletion mutation generated by a targeted disruption in  M. tuberculosis  or  M. bovis  made to date and demonstrates the utility of the specialized transduction system. Moreover, since the parental specialized transducing phage has been shown to infect over 500 clinical  M. tuberculosis  isolates (Jacobs et al., 1987), it should be possible to introduce the RD1 deletion into any  M. tuberculosis  or  M. bovis  isolate of interest. 
     To determine if the RD1 deletion causes an attenuating phenotype in  M. bovis  and  M. tuberculosis , the  M. tuberculosis  H37Rv ΔRD1 was inoculated intravenously into immunocompromised mice possessing the SCID (severe combined immunodeficiency) mutation. Groups of ten mice were injected intravenously with either 2×10 6  wild type or ΔRD1 strain of  M. tuberculosis  and  M. bovis , and three mice per group were sacrificed 24 hours later to verify the inoculation doses. All of the SCID mice infected with the parental  M. tuberculosis  or  M. bovis  strain died within 14 to 16 days post-infection ( FIG. 12A ). In contrast, the SCID mice infected with equal doses of the ΔRD1 strains of  M. tuberculosis  or  M. bovis  were all alive at 25 to 41 days post-infection, demonstrating a highly significant attenuation of the virulence of both strains. It is important to note that BCG-Pasteur kills SCID mice approximately 70 days post-infection ( FIG. 13B ), suggesting that BCG substrains have acquired additional attenuating mutations which are consistent with the deletion analysis of BCG strains (Behr et al., 1999) and the previous failures to restore virulence with the RD1 region (Mahairas et al., 1996). 
     To prove that the attenuation of virulence was due to the RD1 deletion, the  M. tuberculosis  ΔRD1 was transformed with an integrating cosmid, 2F9, containing the RD1 region from  M. tuberculosis  H37Rv 4 . SCID mice were infected as described above and the attenuation for virulence was restored to the parental virulent phenotype ( FIG. 13B ). These results strongly suggest that the genes deleted from the RD1 region contribute to the virulence phenotype. 
     The  M. tuberculosis  ΔRD1 is highly attenuated in immunocompetent BALB/c mice. The virulence of the  M. tuberculosis  ΔRD1 mutant was further assessed by intravenous inoculation of immunocompetent BALB/c mice. While the virulent parent  M. tuberculosis  strain killed the BALB/c mice in 10 to 17 weeks post-infections, 100% of mice were alive at 48 weeks and 43 weeks post-infections in two independent experiments ( FIG. 13C ). 
     While infection with BCG and  M. tuberculosis  ΔRD1 yielded similar survival results in BALB/c mice, there were substantial differences in the growth kinetics in mice. BCG grew in a limited fashion in lungs, liver and spleen in BALB/c mice during the 22 weeks of the experiment ( FIGS. 4B-D ). In contrast, the  M. tuberculosis  ΔRD1 strain grew in a fashion indistinguishable from the parental  M. tuberculosis  H37Rv in all mouse organs for the first 8 weeks. Thereafter, mice infected with the parental  M. tuberculosis  failed to contain the infection leading to mortality. Strikingly, mice infected with the  M. tuberculosis  ΔRD1 showed a definite control over infection resulting in significantly prolonged survival of mice ( FIGS. 4B-D ). 
     Histopathological examination further demonstrated that the mutant was attenuated in virulence compared to the parent strain H37Rv ( FIGS. 5D-F ). In contrast to the rapidly progressive infection with the parent strain, the lung lesions caused by the mutant were maximal in mice examined at 8 weeks post-infection. Consolidating granulomatous pneumonia involved an estimated 25-30% of the lung in these mice. Numerous organisms were demonstrated by acid fast staining. The pneumonia subsequently underwent partial resolution. By 14 weeks, and again, at 22 weeks post-infection, the lungs showed peribronchial and perivascular inflammatory cell accumulations and focal, generally non-confluent, granulomas now with a promiment lymphocytes infiltration. The numbers of acid fast  bacilli  were reduced. Liver lesions consisted of low numbers of scattered granulomas. Spleens were smaller, with persistent granulomas in the red pulp. Mice infected with  M. bovis  BCG showed mild lesions in the lung, liver and spleen at all time points ( FIG. 5G-I ). At longer time intervals post-infection the lesions were fewer in number, and smaller with prominent lymphocytic infiltrations. At 14 weeks post-infection,  M. bovis  BCG was below the level of detection by acid fast staining. In summary, whereas  M. tuberculosis  ΔRD1 initially grew in a manner similar to the parental  M. tuberculosis  H37Rv, this RD1 mutant was limited in the extent of spread of infection, particularly in the lung. This contrasts the extensive and severe damage caused by the parent strain. The subsequent resolving granulomas, localization of the lesions and changes in the composition of the inflammatory cell infiltrations suggested that the mice mounted an effective immune response to combat  M. tuberculosis  ΔRD1 infection and thereby reduced the numbers of viable organisms. 
     Early BCG properties: Altered colonial morphotypes and long-term immunogenicity. While frozen stocks of the original BCG strain do not exist, written records do exist describing the early BCG strains, as Dr. Calmette sent the strains to many laboratories. In a study published in 1929, Petroff and colleagues reported that BCG displayed two distinct colony types (Petroff et al., 1929). One morphotype was a smooth (S) phenotype that was flat and corded (like the parental virulent strain) and the second was a rough and raised (R) phenotype. The  M. tuberculosis  ΔRD1 mutant was generated independently four times and consistently yielded a 20 to 50% mixture of two colonial morphotypes on Middlebrook medium without Tween 80 ( FIG. 3   b ). The distinction of these two types of morphology could be noted even when the colonies were less than two weeks old, as the rough colonies were constricted and elevated with only a small portion of the base of the colony attached to the agar, while the smooth colonies tended to be flattened and spread out. When colonies grew older, e.g. 6 weeks old, the rough colonies remained more constricted compared to those of smooth colonies. The rough colonies exhibited large folds on the surface ( FIG. 3   f - g ), as compared to those of the smooth colonies that exhibited small wrinkles ( FIG. 3   e ). 
     The generation of two distinct colonial morphotypes must be a phenotypic change induced by the deletion of RD1. The morphotypes could not be cloned, as repeated subculturing of either the R or S colonies continued to yield both colonial morphotypes. Moreover, Southern analysis of R and S colonies revealed each morphotype had the same RD1-deleted genotype ( FIG. 3D ). Furthermore, complementation of  M. tuberculosis  ΔRD1 with the RD1 region restored the mutant phenotype back to the homogenous parental S phenotype ( FIG. 3   a - c ). These results suggest that the variable morphotypes resulted directly from the RD1 deletion. It can therefore be postulated that a regulator of colonial morphology is affected by one or more of the deleted genes. 
     The generation of two distinct colonial morphotypes must be a phenotypic change induced by the deletion of RD1. The morphotypes could not be cloned, as repeated subculturing of either the R or S colonies continued to yield both colonial morphotypes. Moreover, Southern analysis of R and S colonies revealed each morphotype had the same RD1-deleted genotype (FIG.  3 D). Furthermore, complementation of  M. tuberculosis  ΔRD1 with the RD1 region restored the mutant phenotype back to the homogenous parental S phenotype ( FIG. 3A-C ). These results suggest that the variable morphotypes resulted directly from the RD1 deletion. It can therefore be postulated that a regulator of colonial morphology is affected by one or more of the deleted genes. 
     One of the hallmark characteristics of BCG is its ability to provide protection against aerosolized challenge with virulent  M. tuberculosis . To test the potential of  M. tuberculosis  ΔRD1 to immunize and protect mice against tuberculous challenge, we used the model of subcutaneous immunization of C57BL/6 mice followed by an aerogenic challenge with virulent  M. tuberculosis  (McGuire et al., 2002). Groups of mice were vaccinated subcutaneously with either 1×10 6  BCG 9 or 1×10 6    M. tuberculosis  ΔRD1. Eight months following vaccination, the mice were all healthy, thereby demonstrating attenuation in a third mouse strain. Vaccinated and unvaccinated mice were aerogenically challenged with 200 CFU of the acriflavin-resistant strain of  M. tuberculosis  Erdman. Twenty-eight days after the challenge, the mice were sacrificed and the bacterial burden in the lungs and spleens were determined (see Table 5). Naive mice served as controls. While the acriflavin-resistant  M. tuberculosis  grew to 6.61±0.13 (log 10  CFU) in lungs of naive mice, both the BCG-vaccinated and  M. tuberculosis  ΔRD1-vaccinated mice exhibited greater than 1 log protection in lungs with CFU values of 5.07±0.10 (p&lt;0.001 relative to controls) and 5.11±0.14 (p&lt;0.001), respectively, detected at the four week time point. The  M. tuberculosis  ΔRD1 also protected against hematogenous spread; CFU values in the spleen were 5.26±0.11 for the controls, 4.00±0.33 (p&lt;0.01) for the  M. tuberculosis  ΔRD1 immunized mice, and 3.85±0.17(p&lt;0.01) for the BCG vaccinated animals. Thus, the  M. tuberculosis  ΔRD1 shares long-term immunogenicity like BCG. 
                     TABLE 5                  Bacterial burden of virulent  M. tuberculosis  in uninoculated mice and       mice inoculated with BCG and H37Rv ΔRD1.                                 Vaccination strain   Lung (log 10 CFU)   Spleen (log10CFU)                       —   6.61 ± 0.13   5.26 ± 0.11           BCG   5.07 ± 0.10***   3.85 ± 0.17**           H37Rv ΔRD1   5.11 ± 0.14***   4.00 ± 0.33**                       **p &lt; 0.01;           ***p &lt; 0.001.            
Discussion
 
     BCG is a mutant of  M. bovis  that was isolated over 94 years ago and characterized for its attenuation for virulence in animals. For over 80 years, BCG has been used as a tuberculosis vaccine having been given to 3 billion humans. It is currently the only anti-tuberculous vaccine available for use in humans, yet its precise attenuating mutations and mechanisms of attenuation have never been determined. Previous studies had identified regions of the  M. bovis  chromosome that were absent from BCG, but present in virulent  M. bovis  and  M. tuberculosis  strains (Mahairas et al., 1996; Gordon et al., 2001). An elegant microarray analysis has also demonstrated that there was only one deletion common to all BCG strains; the authors hypothesized this was the primary attenuating mutation in the original BCG strain isolated by Drs. Calmette and Guerin (Behr et al., 1999). 
     Using a combination of targeted deletion mutagenesis, virulence assays, and complementation analysis, we have been able to unambiguously prove that RD1 is required for virulence for  M. tuberculosis , and by analogy for  M. bovis , for the first time. 
     Moreover, the combination of phenotypes associated with the early BCG strains: i) the attenuation for virulence, ii) the altered colonial morphotypes, and iii) the ability to confer long-term immunogenicity in animals allow us to conclude that the RD1 deletion was the primary attenuating mutation in the original BCG isolate. 
     With regards to the ΔRD1 mutant histology, at 22 weeks post infection, it was noted that the mutant was limited in the extent of the spread of infection, in contrast to the extensive damage caused by the parental strain. Interestingly, Pethe et al. (2001) determined that  M. tuberculosis  needs to bind and/or invade epithelial cells in order to disseminate and cause widespread destruction of the lung, whilst another study reported that pulmonary M cells can act as a portal of entry to the lung for the  tubercle bacilli  (Teitelbaum, 1999). In relation to in vitro analyses, studies utilizing a model of the alveolar barrier, consisting of pneumocytes and monocytes, described how  M. tuberculosis  infection of the pneumocytes resulted in cytolysis, which disrupted the barrier and allowed more efficient translocation of intracellular  bacilli  (Bermudez et al., 2002). 
     Notes 
       1 The following four primers were used to amplify upstream and downstream flanking sequences (UFS and DFS, respectively) for the construction of the RD1 deletion mutants. UFS was amplified using TH201: GGGGGCGCACCTCAAACC (SEQ ID NO:5) and TH202: ATGTGCCAATCGTCGACCAGAA (SEQ ID NO:6). DFS was amplified using TH203: CACCCAGCCGCCCGGAT (SEQ ID NO:7), and TH204: TTCCTGATGCCGCCGTCTGA (SEQ ID NO:8). Recognition sequences for different restriction enzymes were included at the ends of each primer to enable easier manipulation. 
       2 The unmarked deletion mutant of  M. tuberculosis  H37Rv, mc 2 4002, was generated by transformation using a sacB counterselection (Snapper et al., 1988; Pelicic et al., 1996; Pavelka et al., 1999). Specifically, the plasmid pJH508 was created by first cloning UFS into KpnI and XbaI sites, then cloning DFS into EcoRI and HindIII sites of pJH12, a pMV261-derived  E. coli —Mycobacteria shuttle plasmid, to create pJH506 in which UFS and DFS flanked a green fluorescent protein gene (GFPuv, Clonetech) whose expression was driven by the  M. leprae  18Kd promoter. The UFS-gfp-DFS cassette was sub-cloned into the EcoRV site of plasmid pYUB657 to create pJH508. The first homologous recombination involved the identification of hygromycin resistant colonies, resulting from the transformation of  M. tuberculosis  with pJH508. Southern analysis of the NcoI-digested DNA isolated from hygromycin resistant colonies probed with UFS or DFS, confirmed the presence of a single copy of pJH508 inserted into the  M. tuberculosis  genome. The transformant (mc 2 4000) identified was then grown in 7H9 broth to saturation, to allow the second homologous recombination to occur, resulting in recombinants that could be selected by plating the culture on 7H10 plates, supplemented with 3% sucrose. Both Southern analysis and PCR of the DNA isolated from sucrose resistant colonies confirmed the RD1 deletion. 
       3 Specialized transduction is a mycobacteriophage-based method for the delivery of homologous DNA constructs using conditionally replicating shuttle phasmids (Jacobs et al., 1987; Bardarov et al., 1997; Carriere et al., 1997) has been used successfully for  M. tuberculosis  (Glickman et al., 2000, 2001; Raman et al., 2001). Specifically, a transducing phage phAEKO1 was constructed by inserting UFS and DFS into pJSC347, flanking a hygromycin cassette, to create pJH313. pJH313 was digested with PacI and ligated to phAE159, a temperature-sensitive mycobacteriophage derived from TM4. The transduction was performed by growing  M. tuberculosis  to an O.D. 600  of 1.0, washing twice with MP buffer (50 mM Tris pH 7.6, 150 mM NaCl, 10 mM MgCL 2 , 2 mM CaCl 2 ), resuspending into an equal volume of MP buffer and mixing with the transducing phage phAEKO1 at an MOI of 10. The mixtures were incubated at 37° C. overnight, then plated on 7H10 plates supplemented with hygromycin at 50 μg/ml. Hygromycin resistant colonies were analyzed by PCR and Southern analysis, as described above, to confirm the deletion of RD1. 
       4 Complementation analyses was performed using the integration proficient cosmids (Skjot et al., 2000; van Pinxteren et al., 2000) pYUB412 made by S. Bardarov, a library made by F. Bange, and cosmid identified and generously provided by S. T. Cole. 
     EXAMPLE 5 
     Vaccine Efficacy of a Lysine Auxotroph of  M. Tuberculosis    
     In this Example, we describe the in vivo growth phenotype and vaccine efficacy of a lysine auxotrophic mutant of  Mycobacterium tuberculosis  strain H37Rv. An immunization experiment using the mouse model with an aerosol challenge showed that two doses of the  M. tuberculosis  mutant were required to generate protection equivalent to that of the BCG vaccine. 
     Despite the existence of anti-microbial drugs and a widely used vaccine,  Mycobacterium tuberculosis  remains the primary cause of adult death due to a bacterial agent (Dolin et al., 1994). The emergence of multi-drug resistant strains of  M. tuberculosis , the variable efficacy of the current vaccine, the bacille-Calmette and Geurin (BCG), and the HIV pandemic have all contributed to a growing global tuberculosis problem. 
     Several studies have described the development of attenuated auxotrophic strains of BCG and/or  M. tuberculosis  (Guleria et al., 1996); Hondalus et al., 2000; Jackson et al., 1999; Smith et al., 2001). All of these studies utilized single immunization protocols and demonstrated differences in the protective responses thus elicited. In this study, we describe the in vivo growth characteristics of a previously described lysine auxotroph of  M. tuberculosis  H37Rv (Pavelka and Jacobs, 1999), and evaluate the vaccine potential of this mutant by a multiple immunization protocol in a mouse model of the human disease, using an aerosol challenge. 
     Clearance of the  M. tuberculosis  lysine auxotroph in SCID mice. Female SCID mice were bred at the animal facility of the Albert Einstein College of Medicine. The animals were maintained under barrier conditions and fed sterilized commercial mouse chow and water ad libitum. The  M. tuberculosis  strains Erdman, mc 2 3026 (ΔlysA::res) (Id.), and mc 2 3026 bearing pYUB651 (expressing the wild-type lysA gene) were grown in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol, 1×ADS (0.5% bovine serum albumin, fraction V (Roche); 0.2% dextrose; and 0.85% NaCl) or on Middlebrook 7H10 or 7H11 solid medium (Difco) supplemented with 0.2% glycerol and 10% OADC (Becton Dickinson). Culture media for the lysine auxotroph were supplemented with 1 mg/ml of L-lysine (for both liquid and solid media), and 0.05% Tween-80 was also added to solid medium. Liquid cultures were grown in 490 cm 2  roller bottles (Corning) at 4-6 rpm. Plates were incubated for 3-6 weeks in plate cans. All cultures were incubated at 37° C. 
     Titered frozen stocks of the bacteria were thawed and diluted appropriately in phosphate buffered saline containing 0.05% Tween-80 (PBST). The bacterial suspensions were plated at the time of injection to confirm the number of viable bacteria. Intravenous injections were given via a lateral tail vein. At various time points post-injection (24 hours, then once weekly), 3 mice were sacrificed, and the lungs, liver, and spleen removed and homogenized separately in PBST using a Stomacher 80 (Tekmar, Cincinnati, Ohio). The homogenates were diluted in PBST and plated to determine the number of CFU/organ. Note that mice were sacrificed at 24 hours post-injection in order to compare the bacterial colony forming units recovered from the mice with the colony forming units in the suspensions at the time of injection. Thus the bacterial counts reported at time zero actually represent the viable bacteria recovered from the mice at 24-hours post-injection. 
     The lysine auxotrophic strain was cleared from and did not appear to grow in the examined organs of the SCID mice, while the complemented strain multiplied extensively ( FIG. 14 ). Interestingly, the auxotrophic inoculum was cleared from the spleens and lungs but persisted somewhat longer in the liver ( FIG. 14B ). The mice receiving the complemented  M. tuberculosis  mutant died within three weeks of challenge, while the mice given the auxotrophic  M. tuberculosis  mutant did not display any gross organ pathology and survived for at least the duration of the experiment. 
     Two immunizations with the  M. tuberculosis  lysine auxotroph mc 2 3026 are required to match the efficacy of vaccination with BCG-Pasteur. We tested the vaccine potential of the lysine auxotroph mc 2 3026 in the mouse model by means of a virulent aerosol challenge. Female, pathogen-free C57BL/6 mice (Jackson Laboratories, Bar Harbor, Me.) were vaccinated intravenously with ca. 1×10 6  CFU of the  M. tuberculosis  lysine auxotroph or BCG-Pasteur suspended in 0.2 ml PBST. Mice vaccinated with mc 2 3026 were revaccinated at 4 week intervals and the number of viable organisms in the lungs and spleens determined weekly throughout the vaccination period, as described above for the SCID mouse experiments. Five mice were examined at each time point. 
     Immunized mice were challenged 3 months after the initial vaccination. A frozen aliquot of a  M. tuberculosis  Erdman stock was thawed and diluted in PBST to ca. 1×10 6  CFU/ml and 10 ml was introduced into the nebulizer of a Middlebrook aerosol chamber (Glas-Col, Terre Haute, Ind.). The mice were exposed to the infectious aerosol for 30 minutes, inhaling 50-100 CFU into their lungs over this period. Five mice were sacrificed immediately following the challenge period and the lung homogenates were plated to check the amount of the challenge inoculum actually reaching the lungs. Groups of vaccinated and control mice were sacrificed 14, 28, and 42 days later and the lung and spleen homogenates plated to determine the number of viable colony forming units of  M. tuberculosis  Erdman present. Data were analyzed using the Student&#39;s t-test and an analysis of variance between several independent means, using the In Stat Statistics program (GraphPad Software, San Diego). 
     A preliminary experiment demonstrated that a single intravenous immunization of immunocompetent C57BL/6 mice with the  M. tuberculosis  mutant did not generate a significant protective response to the subsequent aerosol challenge with virulent  M. tuberculosis  Erdman. In that experiment, the  M. tuberculosis  auxotroph was rapidly cleared from the mice ( FIG. 15A ), and the single immunization with the auxotroph was insufficient to reduce the bacterial burden in the lungs and spleens relative to a single immunization with BCG ( FIG. 15B ). 
     The failure of the auxotroph to confer protection might have been due to the inability of the mutant to persist long enough, or to synthesize enough antigen to induce an immune response that could significantly restrict the growth of the challenge organisms. One way to circumvent this problem is to give multiple doses of vaccine (Collins, 1991; Homchampa et al., 1992). To this end, mice were intravenously immunized two or three times at four-week intervals with the  M. tuberculosis  lysine auxotroph. In both cases, the vaccine strain was cleared from the lungs and spleens of all the mice at rates similar to that seen with the single immunization experiment ( FIG. 15A ). Three months after the first immunization the mice were challenged with  M. tuberculosis  Erdman by the aerosol route and the bacterial counts in the lungs and spleens were determined and compared to a BCG-Pasteur immunized control, as well as the sham immunized controls. As seen in  FIG. 15C , double immunization with the  M. tuberculosis  lysine auxotroph induced a protective response that was equivalent to that of the BCG control. The reduction in counts in the lung and spleen was equivalent to a 100-fold reduction in bacterial counts compared to the unvaccinated control ( FIG. 15C ). The results from the triple immunization experiment were essentially similar as those from the double immunization experiment described above (data not shown). Furthermore, mice that were immunized with three doses of the  M. tuberculosis  lysine auxotroph and challenged with virulent  M. tuberculosis  Erdman survived at least as long as the BCG-immunized control mice ( FIG. 16 ). 
     Several studies have described the development and vaccine efficacy of attenuated mutant strains of  M. tuberculosis  (Jackson et al., 1999; Hondalus et al., 2000; Smith et al., 2001). The first study reported that a purine auxotroph of  M. tuberculosis  was unable to grow in macrophages and was attenuated for growth in both mice and guinea pigs (Jackson et al., 1999). A guinea pig vaccination experiment determined that a single immunization with the auxotroph allowed the animals to restrict the growth of virulent  M. tuberculosis  in the lungs as well as a single immunization with wild-type BCG, following aerosol challenge. However, the reduction in growth of the challenge organism in the spleen afforded by the auxotroph was not as extensive as that afforded by BCG. Another study reported that a leucine auxotroph of  M. tuberculois  Erdman cannot grow in macrophages and is avirulent to immunocompromised SCID mice (Hondalus et al., 2000). Immunocompetent mice vaccinated once with a  M. tuberculosis  leucine mutant did not significantly restrict the growth of the virulent challenge organism in the lungs or spleen as much as the control mice vaccinated with BCG (Id.). However, the mice immunized with the leucine auxotroph survived as long as the BCG immunized controls and exhibited a decreased histopathology relative to that seen in the non-immunized controls (Id.). A third study showed that  M. tuberculosis  proline and tryptophan auxotrophs were attenuated and a single immunization of mice with either of these mutants afforded protection against an intravenous challenge with virulent  M. tuberculosis , comparable to that for BCG, as indicated by the mean survival times (Smith et al., 2001). In those experiments, mice immunized with pro or trp mutants could restrict the growth of the challenge organisms to the same extent as mice immunized with BCG, although the magnitude of protection in either case ( M. tuberculosis  auxotrophs or BCG) was not as extensive as that seen in the other studies (Id.). 
     In the present study we have demonstrated that a single immunization of mice with the avirulent  M. tuberculosis  lysine auxotroph did not generate an immune response capable of significantly restricting the growth of virulent  M. tuberculosis  Erdman following an aerogenic challenge. However, administration of a second or a third dose of this vaccine increased protection substantially, as measured by the number of viable bacteria per organ, to a level similar to that achieved with single dose of BCG-Pasteur. This level of protection did not seem to be greatly increased by a third dose of vaccine, although the triply immunized mice survived as long as the control mice immunized with a single dose of BCG-Pasteur. Mice that were immunized twice were not followed to determine mean survival time, but comparing the growth curves of the challenge bacteria following the double and triple immunizations, it seems likely that the survival time for the doubly immunized mice would be much the same as that for the triple-immunized mice. 
     The previous studies using  M. tuberculosis  auxotrophs as vaccine strains showed substantial variations in their effectiveness. This variability is likely to be due to a number of factors, including the different  M. tuberculosis  background strains used to construct the mutants, different mouse strains used in the various protection studies, and the different challenge organisms and challenge routes used. There was also considerable variation in the protective efficacy of the different vaccines compared to that observed in controls using BCG immunization. These differences pose a number of questions concerning the best indicators of protection, especially in the long term. Should viable bacterial counts or survival be the primary indicator of protection or should both be given equal weight? The results of this study indicate that more than one immunization with a  M. tuberculosis  lysine auxotroph did generate a significant protective response as indicated by both criteria. We believe it is important that multiple immunization protocols be considered in the further development of attenuated  M. tuberculosis  strains as potential human vaccines. 
     This is the first study demonstrating that a multiple immunization protocol using an auxotroph of  M. tuberculosis  can protect against a highly virulent aerosol challenge compared to that seen for BCG. Since BCG vaccines have shown variable efficacy when tested in humans, an auxotrophic  M. tuberculosis  vaccine might represent an attractive booster vaccine with which to augment childhood BCG immunization. 
     EXAMPLE 6 
     Mutants of  Mycobacterium Tuberculosis  HAving Two Attenuating Mutations are Safe and Provide Protection in Mammals Against Challenge from Virulent Mycobacteria 
     The experiments described in this Example employ materials and methods described in the other Examples. 
     Construction and characterization of  M. tuberculosis  ΔRD1 ΔpanCD (mc 2 6030). A pantothenate auxotroph of  M. tuberculosis  ΔRD1 was generated by specialized transduction and the strain designated mc 2 6030. No CFU were detected on 7H11 when 5×10 10  CFU were plated (repeated twice), suggesting the reversion frequency to be below 10 −11 . 
     SCID mice infected with 1×10 2  CFU H37Rv succumbed to infection in 6 weeks, whereas the mice infected with 1×10 6  mc 2 6030 survived significantly longer with more than 75% of mice surviving for more than 300 days ( FIG. 17A ). Bacteria isolated from mc 2 6030-infected mice before they died were all auxotrophs, confirming that there were no revertants under in vivo conditions. In order to assess the safety of mc 2 6030 in immunocompetent BALB/c mice, we infected mice intravenously with 1×10 6  mc 2 6030 or 1×10 6  of wild-type H37Rv. All mice infected with H37Rv succumbed to infection by 150 days, whereas mice infected with mc 2 6030 survived for more than 300 days ( FIG. 17B ). In an effort to understand the role of immune responses in controlling infection with the pantothenate mutants, we infected immunocompetent C57B1/6 with 1×10 6  CFU of mc 2 6001 (ΔRD1), mc 2 6004 (complementing strain), mc 2 6030 (ΔRD1 ΔpanCD) or wild-type H37Rv. Mice infected with H37Rv and mc 2 6004 showed progressive growth in all the three organs, whereas mice infected with mc 2 6030 showed a drop in growth during the first 3 weeks in the lungs and spleen ( FIG. 18 ). Following 3 weeks of infection, the growth pattern of both mc 2 6001 and mc 2 6030 were identical in the spleen and lungs. Mice immunized subcutaneously with one or two doses of mc 2 6030 demonstrated protection against aerosol challenge with virulent  M. tuberculosis , which was comparable to the protection afforded by BCG vaccination (Table 6). No pantothenate auxotrophs were recovered from spleen or lungs of mice at 1, 2 or 3 months following subcutaneous immunization. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Bacterial burden of virulent  M. tuberculosis  in uninoculated mice and 
               
               
                 mice inoculated with BCG or one or two doses of ΔRD1ΔpanCD. 
               
            
           
           
               
               
               
            
               
                 Experimental Group 
                 Lung CFUs (log 10 ) 
                 Spleen CFUs (log10) 
               
               
                   
               
               
                 Naive 
                 5.99 ± 0.09 
                 4.94 ± 0.06 
               
               
                 ΔRD1ΔpanCD (1 dose) sc 
                 5.22 ± 0.10* 
                 4.04 ± 0.15* 
               
               
                 ΔRD1ΔpanCD (2 doses) sc 
                 4.86 ± 0.14** 
                 3.58 ± 0.11** 
               
               
                 BCG (1 dose) sc 
                 4.79 ± 0.19** 
                 3.73 ± 0.27** 
               
               
                   
               
               
                 *p &lt; 0.01 relative to controls; 
               
               
                 **p &lt; 0.001 relative to controls 
               
            
           
         
       
     
     Construction and characterization of  M. tuberculosis  ΔlysAΔpanCD (mc 2 6020). A pantothenate auxotroph of  M. tuberculosis  ΔlysA was generated by specialized transduction and the strain designated mc 2 6020. No CFU were detected on 7H11 when 5×10 10  CFU were plated, suggesting the reversion frequency to be below 10 −11 . This double mutant is auxotrophic for both lysine and pantothenate. SCID mice infected with 1×10 2  CFU H37Rv succumbed to infection in 6 weeks, whereas the mice infected with 1×10 6  mc 2 6020 survived for more than 400 days with no mortality. In order to assess the safety and growth kinetics of mc 2 6020 in immunocompetent BALB/c mice, we infected mice intravenously with 1×10 6  mc 2 6020 or 1×10 6  of wild-type H37Rv. All mice infected with H37Rv succumbed to infection by 150 days, whereas mice infected with mc 2 6020 survived for more than 400 days. After 3 weeks following intravenous infection, no colonies of mc 2 6020 could be recovered from spleen, liver or lungs of infected mice. Interestingly, mice immunized subcutaneously with one or two doses of mc 2 6020 demonstrated protection against aerosol challenge with virulent  M. tuberculosis , which was comparable to the protection afforded by BCG vaccination (Table 7). No pantothenate and lysine requiring auxotrophs were recovered from spleen or lungs of mice at 1, 2 or 3 months following subcutaneous immunization. Other studies established that both mc 2 6020 and mc 2 6030 protects the a level of protection of mice against TB equivalent to the protection afforded by BCG ( FIG. 19 ). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Bacterial burden of virulent  M. tuberculosis  in uninoculated mice and 
               
               
                 mice inoculated with BCG or one or two doses of mc 2 6020 
               
               
                 (ΔlysAΔpanCD) sc or one dose of mc 2 6020 iv. 
               
            
           
           
               
               
               
            
               
                 Experimental group 
                 Lung CFUs (log 10 ) 
                 Spleen CFUs (log 10 ) 
               
               
                   
               
               
                 naive 
                 6.03 ± 0.05 a   
                 4.84 ± 0.27 
               
               
                 BCG (1 dose) sc 
                 4.76 ± 0.19*** 
                 3.95 ± 0.18* 
               
               
                 mc 2 6020 (1 dose) sc 
                 5.05 ± 0.06*** 
                 4.02 ± 0.11* 
               
               
                 mc 2 6020 (2 doses) sc 
                 5.09 ± 0.05*** 
                 4.06 ± 0.27 
               
               
                 mc 2 6020 (1 dose) iv 
                 5.06 ± 0.11*** 
                 4.00 ± 0.15* 
               
               
                   
               
               
                   a Mean ± SEM 
               
               
                 p &lt; 0.001 =***; 
               
               
                 p &lt; 0.05 =* 
               
            
           
         
       
     
     These data clearly demonstrate the safety and immunogenicity of these two double mutants of  M. tuberculosis  in mice. 
     The double deletion mutant mc 2 6030 (ΔRD1ΔpanCD) immunizes and protects SCID mice from aerosolized  M. tuberculosis  challenge. The double deletion mutants were safer than BCG in SCID mice, where all of the SCID mice died before 100 days when inoculated with BCG, 100% and 25% of the mice survived inoculation with mc 2 6020 and mc 2 6030, respectively ( FIG. 20 ). 
     In view of the above, it will be seen that the several advantages of the invention are achieved and other advantages attained. 
     As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. 
     All references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.