Manipulation of BCG vaccine: a double-edged sword.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG), an attenuated vaccine derived from M. bovis, is the only licensed vaccine against tuberculosis (TB). Despite its protection against TB in children, the protective efficacy in pulmonary TB is variable in adolescents and adults. In spite of the current knowledge of molecular biology, immunology and cell biology, infectious diseases such as TB and HIV/AIDS are still challenges for the scientific community. Genetic manipulation facilitates the construction of recombinant BCG (rBCG) vaccine that can be used as a highly immunogenic vaccine against TB with an improved safety profile, but, still, the manipulation of BCG vaccine to improve efficacy should be carefully considered, as it can bring in both favourable and unfavourable effects. The purpose of this review is not to comprehensively review the interaction between microorganisms and host cells in order to use rBCG expressing M. tuberculosis (Mtb) immunodominant antigens that are available in the public domain, but, rather, to also discuss the limitations of rBCG vaccine, expressing heterologous antigens, during manipulation that pave the way for a promising new vaccine approach.

Synthesis and antitubercular activities of bis-glycosylated diamino alcohols.

Conjugate addition of diamines to glycosyl olefinic esters 1a and 1b followed by reduction of resulting bis-glycosyl beta-amino esters (2-7 and 14-19) with lithium aluminium hydride led to the respective glycosyl amino alcohols (8-13 and 20-25) in moderate to good yields. All the compounds were evaluated for antitubercular activity against Mycobacterium tuberculosis H(37)Ra and H(37)Rv. Few of the compounds exhibited antitubercular activity with MIC as low as 6.25-3.12microg/mL in virulent and avirulent strains. Compound 13 was found to be active against MDR strain and showed mild protection in mice.

Synthesis and antimycobacterial activities of glycosylated amino alcohols and amines.

Reduction of glycosyl beta-amino esters (6-14 and 25-30) with lithium aluminum hydride resulted in glycosyl amino alcohols (15-23 and 31-36) in good yields. However, reductive amination of glycosyl aldehydes (1-3) with different amines in presence of sodium borohydride resulted in good to moderate yields of glycosyl amines (37-41). All the compounds were evaluated for antitubercular activity against Mycobacterium tuberculosis H37Ra and H37Rv. Compounds 18, 21, 35 and 36 exhibited antitubercular activities with MIC ranging from 6.25 to 3.12 microg ml(-1).

Synthesis of galactopyranosyl amino alcohols as a new class of antitubercular and antifungal agents.

The galactopyranosyl amino alcohols (3-16) were synthesised by regioselective oxirane ring opening of compound 2 with variety of amines and screened for antitubercular and antifungal activities. One of the compounds (16) showed potent activity against Mycobacterium tuberculosis H37 Rv in vitro and also displayed activity in MDR TB. The compound (16) was found to be superior to ethambutol clinically used anti TB drug in in vitro screen.

Protection by live Mycobacterium habana vaccine against Mycobacterium tuberculosis H37Rv challenge in mice.

BACKGROUND & OBJECTIVES: In recent years the efficacy of BCG vaccine against tuberculosis has been questioned and there is no alternative vaccine available. Several strategies are being applied to get a satisfactory vaccine. Two approaches are generally considered: the subunit vaccines and the whole cell vaccines. The objective of this investigation was to evaluate an avirulent mycobacteria, Mycobacterium habana, as a whole cell vaccine to protect mice from infection of M. tuberculosis H37Rv. METHODS: AKR and immunocompromised SJL/J mice were immunized with live M. habana vaccine. These mice were challenged with M. tuberculosis H37Rv eight weeks later along with unimmunized control mice. Protection by M. habana vaccine was measured through several parameters, which included survival of challenged mice, dissemination of challenge strain and histopathology of lung tissues. RESULTS: M. habana vaccinated animals were healthier than the unvaccinated mice after challenge with M. tuberculosis and survived with significant increase in mean survival time. The viable count of challenge strain was at least 100-fold less in vaccinated mice than the control mice. The lung tissues in unvaccinated mice showed marked bronchopneumonia with clusters of acid fast bacilli, whereas vaccinated mice showed small areas of damage and evidence of protection subsequently. INTERPRETATION & CONCLUSION: It may be concluded from the evidence presented here that mice vaccinated with M. habana were protected from challenge with M. tuberculosis in both normal and immunocompromised states.

Synthesis and antimycobacterial activity of 3,5-disubstituted thiadiazine thiones.

A series of 3,5-disubstituted thiadiazine thiones (4-24) have been synthesized by reaction of primary amines with carbon disulphide followed by cyclocondensation of the resulting intermediate with formaldehyde and primary amines or amino acids. The compounds were screened for antitubercular activity in vitro against Mycobacterium tuberculosis H37Rv. Three compounds 4, 12 and 18 showed antimycobacterial activity with MIC 12.5 microg/mL. Compound 4, was tested in vitro against five multidrug resistant (MDR) strains of M. tuberculosis and was found to be active. Compound 4 also exhibited activity in vivo. While all the mice died in the untreated group, the mean survival time (MST) of the compound treated mice was enhanced, 33% mice were surviving in treated group and the load of bacilli in the lung was considerably less in the compound treated group than in the untreated control group.

Synthesis and bioevaluation of glycosyl ureas as alpha-glucosidase inhibitors and their effect on mycobacterium.

Glycosyl amino esters (2-13) on reaction with different isocyanates resulted in quantitative conversion to glycosyl ureas (14--32). Few of the selected ureas (15-20, 22-28, 30 and 32) on cyclative amidation with DBU/TBAB/4 A MS gave respective dihydropyrimidinones in fair to good yields (33-47). The compounds were screened for alpha-glucosidase inhibitory activity and two (19 and 23) of them showed strong inhibition against rat intestinal alpha-glucosidase. The compounds were also screened against Mycobacterium aurum, however, only one (19) of them exhibited marginal antitubercular activity.

Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach.

The in vivo induced antigen technology (IVIAT)(1) has been used for the identification of open reading frames (ORFs) which could be possible therapeutic targets. A recombinant lambdagt11:: Mycobacterium tuberculosis H37Rv expression library was screened with pooled TB patient sera preabsorbed with in vitro grown M. tuberculosis H37Rv. Preabsorption of pooled TB patient sera allowed identification of antigens specifically expressed or upregulated during infection and growth in vivo. Six ORFs were identified, of which four (rv0287, rv2402, rv3878 and rv1045) were of hypothetical functions. Rv0287 is a probable regulatory protein. Rv3878 is present uniquely in M. tuberculosis H37Rv and is a part of RDI deletion region of M. bovis BCG, which includes esat 6 region. This could be exploited as a tool for diagnosis. Two ORFs were assigned function solely on the basis of homology, dnaQ (rv3711c) and lpdA (rv3303c). dnaQ codes for the epsilon subunit of DNA polymerase III, which is responsible for the proofreading activity of the complex. lpdA codes for dihydrolipoamide dehydrogenase, which is a part of many multienzyme complexes such as pyruvate dehydrogenase, keto-acid dehydrogenase and alpha-ketoglutarate dehydrogenase. These two enzymes appear to be potential targets for drug development.

Synthesis of glycosylated beta-amino acids as new class of antitubercular agents.

A series of glycosylated beta-amino acids was prepared and evaluated against Mycobacterium tuberculosis, M. avium, M. fortuitum and M. smegmatis. The compounds were designed to mimic the enzyme D-alanine racemase and glycosyl transferase involved in the biosynthesis of essential cell wall peptidoglycan and arabinogalactan. Though most of the compounds exhibited little activity, however, one showed significant activity against all the strains in cell culture and activity was confirmed by BACTEC method.

Bioluminescent Mycobacterium aurum expressing firefly luciferase for rapid and high throughput screening of antimycobacterial drugs in vitro and in infected macrophages.

The slow growth and highly infectious nature of Mycobacterium tuberculosis is a limiting factor in its use as test organism in high throughput screening for inhibitory compounds. To overcome these problems, use of surrogate strains and reporter genes have been considered. In this study, we have investigated the application of a fast growing nonpathogenic M. aurum expressing firefly luciferase in rapid screening of antituberculosis compounds in vitro and in infected macrophages using bioluminescence assay. The assay is based on luminescence determination using luciferin as substrate. Inhibition of bioluminescence was obtained with frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their reported MICs. Inhibition could be observed as early as 2 h in vitro and within 24 h in infected macrophages. The system can reliably be used in high throughput screening.

Protective antigens of Mycobacterium habana are distributed between peripheral and integral compartments of plasma membrane: a study in experimental tuberculosis of mouse.

Mycobacterium habana, a cultivable nonpathogenic mycobacterium provides appreciable resistance in mouse against M. tuberculosis infection. This study is aimed at identification and characterization of protective antigens of M. habana. Protective potential of antigens of cell wall (CW), cell membrane (CM), cytosol (CS) and peripheral and integral compartments of the membrane fraction of M. habana was explored against experimental tuberculosis in mouse. Peripheral and integral membrane proteins were characterized by SDS-PAGE and differential staining with silver and periodic acid. Results reveal that protective antigens are distributed in both peripheral and integral membrane compartments though such effect is dominant in the former. Polysaccharide staining showed that LAM, LM and PIMs have a preference for the detergent phase. Peripheral and integral compartments constitute, respectively, 68 and 31% of the total membrane protein.

Genetic rearrangements leading to disruption of heterologous gene expression in mycobacteria: an observation with Escherichia coli beta-galactosidase in Mycobacterium smegmatis and its implication in vaccine development.

Different mycobacteria carrying cloned genes for heterologous protective antigens have been proposed as vaccine vehicles. In this study, the stability of the expression of beta-galactosidase was studied in Mycobacterium smegmatis using integrative (pMV361::lacZ) and replicative (pMV261::lacZ) vectors. Recombinant M. smegmatis forms blue colonies on X-gal plates. Occasional white mutants encountered while plating on X-gal plates were genetically analysed. The loss of lacZ phenotype was due to insertion of an IS element in lacZ gene of integrative vector whereas in case of replicative vectors, loss of lacZ phenotype was due to deletions of different sizes in the lacZ gene and the Phsp60 promoter region. The frequency of such events was rare, 1.7 x 10(-5) in integrative vector and 2 x 10(-3) in the case of replicative vector. The integrative vector seemed more stable in terms of expression of foreign genes in mycobacteria. Hence, the rearrangements reported in the present study warrant serious consideration before implementing mycobacteria as recombinant vaccines.

Differential expression of proteins during healing of cutaneous wounds in experimental normal and chronic models.

Recent studies have demonstrated growth factors and other cellular proteins as being important in the healing process. In this study, we have investigated the differential expression of proteins in wound tissues of normal and chronic animal models. Proteins were identified by specific antibodies, partial N-terminal amino acid sequence, and molecular weight homology. In normal wound tissues de novo synthesis of a heat shock protein, platelet derived growth factor (PDGF), and fibroblast growth factor (FGF) was induced within 24 h of skin injury. Proteins resembling vascular endothelial growth factor, receptors for PGDF, FGF, and epidermal growth factor, were synthesized. The elevated synthesis declined to a basal level in 7 to 14 days after skin injury which coincided with healing of wounds. These changes occurred only in wound site tissues but not in distal tissues. In contrast, the chronic wounds presented a different picture. The expressions of these proteins were either delayed or inhibited. This suggested the role of these proteins during normal and chronic wound healing. The proteins which were down regulated in chronic wounds may be used in the management of wounds and exploited as targets for therapeutic development.

Green fluorescent protein as a reporter in rapid screening of antituberculosis compounds in vitro and in macrophages.

The development of new drugs against Mycobacterium tuberculosis is impeded by slow growth and highly infectious nature of the organism that warrants the need to work under highly stringent biosafety conditions. These problems can be overcome by use of reporter genes and surrogate strains. A strain of rapidly growing M. aurum has been recommended as test organism to screen inhibitors of mycobacteria to preselect compounds for progression into testing against M. tuberculosis. We have investigated the application of recombinant M. aurum expressing green fluorescent protein in rapid screening of antituberculosis compounds in vitro and in infected macrophages. Recombinant M. aurum[pGFM-11] expressing green fluorescent protein was constructed. The assay is based on measurement of fluorescent intensity at 509 nm. A good correlation was found between fluorescence and growth. Fluorescence of recombinant M. aurum was inhibited in vitro within 8 to 24 h by frontline antimycobacterial drugs at their reported MICs whereas inhibition in infected macrophages was observed in 72 h. Therefore green fluorescent reporter system provides a convenient screen to test antimycobacterial compounds that are active in vitro and within infected macrophages.

Diagnosis of tuberculous meningitis by detection of antigen and antibodies in CSF and sera.

OBJECTIVE: To evaluate diagnostic potential of three immunological tests, namely, detection of H37Rv antigen of M. Tuberculosis in CSF, detection of antibodies (IgG) against H37Rv in CSF and detection of antibodies (IgG) against H37Rv in serum for diagnosis of tuberculous meningitis in children. SUBJECTS: 50 children diagnosed as patients of tuberculous meningitis were included as cases and 48 children with CNS diseases of nontubercular etiology [pyogenic meningitis (n = 31), encephalitis (n = 10), seizure disorder of unknown etiology (n = 5), brain tumor (n = 2)] served as controls. METHODS: H37Rv antigen of M. tuberculosis was detected in CSF by Dot ELISA, and antibodies (IgG) against H37Rv in CSF and serum were detected by Plate ELISA. RESULTS: Detection of H37Rv antigen in CSF was the most sensitive (90%) and specific (95.83%) with positive and negative predictive values of 95.74% and 90.19%, respectively, followed by detection of antibodies in CSF (sensitivity-74%, specificity-89.58%, positive predictive value-88.10%, negative predictive value-76.78%). Detection of antibodies in serum had low sensitivity (50%), specificity (91.67%), positive predictive value (86.21%) and negative predictive value (63.76%). CONCLUSIONS: Detection of antigen in CSF is a rapid, sensitive and specific test for diagnosis of tuberculous meningitis in children. Detection of antibody in CSF may be useful in some cases but needs further evaluation. Detection of antibody in serum does not appear to be useful for diagnosis of tuberculous meningitis.

Recombinant Mycobacterium aurum expressing Escherichia coli beta-galactosidase in high throughput screening of antituberculosis drugs.

Mycobacterium aurum is considered a surrogate of M. tuberculosis and recently has been proposed as test organism in high throughput screening of antituberculosis drugs (3). In this investigation, we suggest use of a recombinant M. aurum expressing E. coli lacZ gene, in which beta-galactosidase production is the reporter system as recently reported by us (6). The assay is based on production of beta-galactosidase in presence of drugs during growth. Enzyme production was inhibited within 4 h by frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their MICs. The assay could be performed conveniently in 96-well microtiter plate format.

Screening of TnphoA mutants of Vibrio cholerae O139 for identification of antigens involved in colonisation.

A new serogroup of Vibrio cholerae non-O1, designated as O139, has emerged causing cholera-like disease among adults. Laboratory and field studies clearly show that there is no cross-protection between O1 and O139 pathogenic strains. Since colonisation of the intestine is a most important step in the pathogenesis of cholera caused by O1 strains and colonising antigens are known to be protective, investigation of the colonising antigens of O139 strain was initiated. By TnphoA mutagenesis, mutants were generated with insertions in the genome encoding membrane spanning or secretory proteins. Screening of the mutants for adherence to rabbit intestinal surface and colonisation in 5-day-old mice resulted in the identification of mutant clones, which were less adhesive than was the wild-type parent strain and which could not efficiently colonise the gut. Such non-colonising strains were attenuated in virulence. Analysis of the proteins by SDS-PAGE revealed that the non-colonising mutants did not express a 40-kDa outer-membrane protein.

beta-Galactosidase reporter system in mycobacteria and its application in rapid antimycobacterial drug screening.

Pathogenic mycobacteria are generally slow growing organisms and it takes several weeks to evaluate inhibitors of growth. Therefore, for rapid screening of the inhibitors of mycobacterial growth, a beta-galactosidase reporter system has been described which utilises a recombinant Mycobacterium smegmatis mc(2)155 expressing E. coli lacZ gene as the test organism. The assay is based on production of beta-galactosidase in presence of drugs during growth. A correlation between beta-galactosidase production and colony forming ability of mycobacteria was obtained. beta-galactosidase production was inhibited within 6 h by front line standard antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, pyrazinamide and ofloxacin at their reported MICs. The assay was performed on mycobacterial cells permeabilized with chloroform and sodium dodecyl sulfate.

Evaluation of different subcellular fractions of Vibrio cholerae O139 in protection to challenge in experimental cholera.

Various cellular fractions of Vibrio cholerae O139 were prepared and evaluated in the rabbit ileal loop model of experimental cholera for identification of the protective antigen(s) relevant for vaccine development. Lipopolysaccharides (LPS) and capsular polysaccharides (CPS) of O139 strains and its cell surface, membrane and cytosolic fractions were assayed for antibacterial immunity, whereas the cholera toxin was examined for antitoxic immunity. The lipopolysaccharides, membrane fraction and cholera toxin induced moderate protection, however there was a significant synergistic effect when cholera toxin was combined with membrane proteins or lipopolysaccharides. The O139 strains strongly resembled O1 strains in the profile of proteins and immunological cross reactivity, yet there was no cross protection. The results warrant further investigation of the pathogenesis of O139 strains and identify the critical somatic antigens relevant to protection.