Immunogenicity of orally-delivered lipid-formulated BCG vaccines and protection against Mycobacterium tuberculosis infection

Lipid formulations containing BCG strains Danish 1331 or Moreau (Rio de Janeiro) were trialled as oral vaccines in rodent models. In mice, oral-delivery of either strain resulted in BCG colonisation of the alimentary tract lymphatics and induction of gamma-interferon responses. In guinea pigs, both strains provided pulmonary protection against Mycobacterium tuberculosis aerosol challenge, as shown by significantly reduced bacterial loads and lung:body weight ratios. Lipid-formulated BCG provided superior protection against M. tuberculosis over unformulated orally-delivered BCG (Moreau), and equivalent protection to sub-cutaneous BCG (Danish) immunisation. Oral-delivery of lipid-formulated BCG may offer a practical alternative to parenteral-route BCG vaccination.     

Vaccine efficacy of Mycobacterium bovis BCG against Mycobacterium sp. infection in amberjack Seriola dumerili

Mycobacteriosis, caused by the intracellular parasitism Mycobacterium sp., causes economic damages to aquaculture production in Japan, particularly in seriola fish production. Antibiotics are not effective against Mycobacterium sp. and so a potent vaccine is needed. We previously reported that BCG vaccine (Mycobacterium bovis BCG) induces adaptive immunity against Mycobacterium sp. in Japanese flounder, Paralichthys olivaceus. In a phylogenetic tree, the genes for a major antigen, the Ag85 complex, in Mycobacterium sp. TUMSAT-Msp001 are closely related to homologues in Mycobacterium ulcerans. M. bovis BCG was detected until 7 days post-injection at the injection site (muscle) and 28 days post-vaccination in spleen. Cumulative mortality of amberjack, Seriola dumerili vaccinated intramuscularly (i.m.) and intraperitoneally (i.p.) with M. bovis BCG was 32.3% and 59.5% respectively, at 24 days post-infection of Mycobacterium sp., compared to 97.8% in PBS-injected fish. The bacterial counts of Mycobacterium sp. in spleen of both i.m.-and i.p.-vaccinated fish (6.2 × 10³ and 1.3 × 10⁴ CFU/mg tissue, respectively) at 20 days post-infection were significantly lower (P < 0.01) than those of PBS-injected fish (8.0 × 10⁶ CFU/mg). Furthermore, Immersion challenge with Mycobacterium sp. TUMSAT Msp-001 showed 50% RPS value in BCG i.m.-vaccinated fish at the end of the experiment. These results support our previous study using Japanese flounder and suggest that BCG vaccine is also effective against Mycobacterium sp. infection in amberjack.     

BCG induces protection against Mycobacterium tuberculosis infection in the Wistar rat model

Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccination in this model. Upon Mtb challenge, BCG vaccinated rats controlled growth of the bacilli earlier than unvaccinated rats. Histopathology analysis of infected lungs demonstrated a reduced number of granulomatous lesions and lower parenchymal inflammation in vaccinated animals. Vaccine-mediated protection correlated with the rapid accumulation of antigen specific CD4(+) and CD8(+) T cells in the infected lungs. Immunohistochemistry further revealed higher number of CD8(+) cells in the pulmonary granulomas of vaccinated animals. Evaluation of pulmonary immune responses in vaccinated and Mtb infected rats by real time PCR at day 15 post-challenge showed reduced expression of genes responsible for negative regulation of Th1 immune responses. Thus, early protection observed in BCG vaccinated rats correlated with a similarly timed shift of immunity towards the Th1 type response. Our data support the importance of (i) the Th1-Th2 balance in the control of mycobacterial infection and (ii) the value of the Wistar rats in understanding the biology of TB.     

Enzymatic and antigenic analyses of strains of Mycobacterium bovis,M. bovis BCG,and M. tuberculosis

Strains of Mycobacterium bovis, M. bovis BCG, and M. tuberculosis, including a so-called Canetti strain, were analyzed by means of two-dimensional immunoelectrophoresis (2D-IE), 2D-IE combined with enzyme staining, and multilocus enzyme electrophoresis (MEE). The results demonstrated a close antigentic and enzymatic resemblance among all the strains tested, even though the BCG strains could be divided into two groups based on the presence of one precipitinogen. Eight of the precipitinogens were shown to correspond to enzymes in M. bovis BCG and 10 in M. tuberculosis. Thus, catalase, isocitrate dehydrogenase, malate dehydrogenase, peroxidase, and several others were identified. By means of MEE the strains of M. tuberculosis, M. bovis, and M. bovis BCG could be differentiated. The analyses further indicated that the M. tuberculosis strain Canetti was more closely related to M. bovis than to M. tuberculosis.     

M.tuberculosis Mutants Lacking Oxygenated Mycolates Show Increased Immunogenicity and Protective Efficacy as Compared to M. bovis BCG Vaccine in an Experimental Mouse Model

The existing vaccine against tuberculosis (M. bovis BCG) exerts some protection against the extrapulmonary forms of the disease, particularly in young children, but is not very effective against the pulmonary form of TB, which often results from the reactivation of a latent M. tuberculosis (M.tb)infection. Among the new approaches in TB vaccine development, live attenuated M.tb mutants are a promising new avenue. Here we report on the vaccine potential of two highly attenuated M.tb mutants, MGM1991 and M.tbhma::hyg (HMA), lacking all oxygenated mycolates in their cell wall. In C57BL/6 mice, stronger Th1 (IFN-γ, IL-2 and TNF-α) and IL-17 responses could be induced following subcutaneous vaccination with either of the two mutants, than following vaccination with M. bovis BCG. Significantly more mycobacteria specific IFN-γ producing CD4⁺ and particularly CD8⁺ T cells could be detected by intracellular cytokine staining in mice vaccinated with the M.tb mutants. Finally, vaccination with either of the two mutants conferred stronger protection against intratracheal M.tb challenge than vaccination with BCG, as indicated by reduced bacterial replication in lungs at 4 to 12 weeks after challenge. Protection against M. tb dissemination, as indicated by reduced bacterial numbers in spleen, was comparable for both mutants to protection conferred by BCG.     

Antibodies against BCG antigen 60 in mycobacterial infection.

A sensitive specific radioimmunoassay was developed to measure antibodies against BCG antigen 60, a prominent antigenic component of BCG bacilli which cross-reacts with similar components in many mycobacterial species including Mycobacterium leprae and M tuberculosis. A lepromatous serum pool had anti-BCG-60 activity with a titre of 10(5) and the tuberculoid pool a titre of 10(4). Testing of individual sera showed striking variations within groups of patients with lepromatous and tuberculoid leprosy. In five of the 20 tuberculoid leprosy sera the anti-BCG-60 activity was above the median for the lepromatous group. The current view that antibody formation against mycobacterial antigens is very low in tuberculoid leprosy thus no longer appears to be tenable. Sera from eight patients with active pulmonary tuberculosis also showed a striking variation in anti-BCG-60 content, and the median value of this group was even higher than in those with lepromatous leprosy.     

Loperamide exerts a direct bactericidal effect against M. tuberculosis,M. bovis,M. terrae and M. smegmatis

Tuberculosis (TB) is caused by Mycobacterium tuberculosis. TB is highly prevalent, characterized by the constant occurrence of drug-resistant cases, and confounded by the incidence of respiratory disease caused by non-tuberculous mycobacteria (NTB). Expanding the spectrum of drugs for the treatment of TB is indispensable. Loperamide, an antidiarrhoeal drug, enhances immune-driven antimycobacterial activity, and we aimed to evaluate its bactericidal activity against M. tuberculosis, Mycobacterium bovis BCG, Mycobacterium terrae and Mycobacterium smegmatis. Loperamide exhibited an inhibitory effect against all mycobacterial species tested, with MICs of 100 and 150 μg ml⁻¹. Thus, loperamide is a mycobactericidal drug with potential as adjunctive therapy for TB and NTB infections.     

Oxadiazolone derivatives,new promising multi-target inhibitors against M. tuberculosis

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc²6230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC₅₀, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.     

Protective effect of breast feeding against infection.

OBJECTIVE--To assess the relations between breast feeding and infant illness in the first two years of life with particular reference to gastrointestinal disease. DESIGN--Prospective observational study of mothers and babies followed up for 24 months after birth. SETTING--Community setting in Dundee. PATIENTS--750 pairs of mothers and infants, 76 of whom were excluded because the babies were preterm (less than 38 weeks), low birth weight (less than 2500 g), or treated in special care for more than 48 hours. Of the remaining cohort of 674, 618 were followed up for two years. INTERVENTIONS--Detailed observations of infant feeding and illness were made at two weeks, and one, two, three, four, five, six, nine, 12, 15, 18, 21, and 24 months by health visitors. MAIN OUTCOME MEASURE--The prevalence of gastrointestinal disease in infants during follow up. RESULTS--After confounding variables were corrected for babies who were breast fed for 13 weeks or more (227) had significantly less gastrointestinal illness than those who were bottle fed from birth (267) at ages 0-13 weeks (p less than 0.01; 95% confidence interval for reduction in incidence 6.6% to 16.8%), 14-26 weeks (p less than 0.01), 27-39 weeks (p less than 0.05), and 40-52 weeks (p less than 0.05). This reduction in illness was found whether or not supplements were introduced before 13 weeks, was maintained beyond the period of breast feeding itself, and was accompanied by a reduction in the rate of hospital admission. By contrast, babies who were breast fed for less than 13 weeks (180) had rates of gastrointestinal illness similar to those observed in bottle fed babies. Smaller reductions in the rates of respiratory illness were observed at ages 0-13 and 40-52 weeks (p less than 0.05) in babies who were breast fed for more than 13 weeks. There was no consistent protective effect of breast feeding against ear, eye, mouth, or skin infections, infantile colic, eczema, or nappy rash. CONCLUSION--Breast feeding during the first 13 weeks of life confers protection against gastrointestinal illness that persists beyond the period of breast feeding itself.     

Capreomycin is active against non-replicating M. tuberculosis

Background

Eradication of Helicobacter pylori is an important objective in overcoming gastric diseases. Many regimens are currently available but none of them could achieve 100% success in eradication. Eugenol and cinnamaldehyde that are commonly used in various food preparations are known to possess antimicrobial activity against a wide spectrum of bacteria.

Aim

The present study was performed to assess the in vitro effects of eugenol and cinnamaldehyde against indigenous and standard H. pylori strains, their minimum inhibitory concentrations (MICs) and time course lethal effects at various pH.

Methods

A total of 31 strains (29 indigenous and one standard strain of H. pylori ATCC 26695, one strain of E. coli NCIM 2089) were screened. Agar dilution method was used for the determination of drug sensitivity patterns of isolates to the commonly used antibiotics and broth dilution method for the test compounds.

Results

Eugenol and cinnamaldehyde inhibited the growth of all the 30 H. pylori strains tested, at a concentration of 2 μg/ml, in the 9th and 12th hours of incubation respectively. At acidic pH, increased activity was observed for both the compounds. Furthermore, the organism did not develop any resistance towards these compounds even after 10 passages grown at sub-inhibitory concentrations.

Conclusion

These results indicate that the two bioactive compounds we tested may prevent H. pylori growth in vitro, without acquiring any resistance.     

Protective effects of supplemental vitamin E against infection.

Vitamin E supplementation (dl-alpha-tocopheryl acetate except where noted) in excess of requirement significantly increased humoral immune response or disease resistance. Mice immunized with sheep red blood cells or tetanus toxoid and fed the supplemental vitamin demonstrated increased plaque-forming cells (PFC) and hemagglutinin (HA) titers. A vitamin E deficiency resulted in decreased PFC and little IgG which was partially corrected by N,N-diphenyl-p-phenylenediamine but not as effectively as by vitamin E. Hens immunized with Brucella abortus and fed different levels of the vitamin produced chicks with increased passive immunity; a biphasic antibody response to the level of the vitamin fed was noted. Vitamin E fed to nonimmunized hens was found to significantly increase the primary immune response of their immunized chicks. Feeding dl-alpha-tocopheryl acetate to guinea pigs immunized with Venezuelan equine encephalomyelitis virus resulted in no increased immunity. Injecting this form of the vitamin resulted in severe tissue reaction. However, injecting dl-alpha-tocopheryl significantly improved hemagglutinin inhibition titers. Chicks and turkeys infected with Escherichia coli and fed supplemental vitamin E had reduced mortality and increased HA titers. Sheep fed vitamin E and challenged with Chlamydia had improved weight gains and no detectable Chlamydia.     

Protective efficacy of different cell-wall fractions ofMycobacterium tuberculosis

Immunization with various cell-wall fractions ofM. tuberculosis H₃₇Ra, progressively depleted of lipids (cell-wall-insoluble fraction; CWIF), soluble proteins (cell-wall core; CWC), mycolic acids and arabinogalactans (cell-wall-protein-peptidoglycan complex; CW-PPC) elicited significant levels of both humoral and cell-mediated immune response. Mice immunized with these fractions, when challenged with an LD₅₀ dose ofM. tuberculosis H₃₇Rv, exhibited significant protection as revealed by high survival rates and decreased bacterial load in lungs, liver and spleen, as compared to nonimmunized animals.     

Activity of Trifluoperazine against Replicating, Non-Replicating and Drug Resistant M. tuberculosis

Trifluoperazine, a knowm calmodulin antagonist, belongs to a class of phenothiazine compounds that have multiple sites of action in mycobacteria including lipid synthesis, DNA processes, protein synthesis and respiration. The objective of this study is to evaluate the potential of TFP to be used as a lead molecule for development of novel TB drugs by showing its efficacy on multiple drug resistant (MDR) Mycobacterium tuberculosis (M.tb) and non-replicating dormant M.tb. Wild type and MDR M.tb were treated with TFP under different growth conditions of stress like low pH, starvation, presence of nitric oxide and in THP-1 infection model. Perturbation in growth kinetics of bacilli at different concentrations of TFP was checked to determine the MIC of TFP for active as well as dormant bacilli. Results show that TFP is able to significantly reduce the actively replicating as well as non-replicating bacillary load. It has also shown inhibitory effect on the growth of MDR M.tb. TFP has shown enhanced activity against intracellular bacilli, presumably because phenothiazines are known to get accumulated in macrophages. This concentration was, otherwise, found to be non-toxic to macrophage in vitro. Our results show that TFP has the potential to be an effective killer of both actively growing and non-replicating bacilli including MDR TB. Further evaluation and in vivo studies with Trifluoperazine can finally help us know the feasibility of this compound to be used as either a lead compound for development of new TB drugs or as an adjunct in the current TB chemotherapy.     

Overexpression of inhA,but not kasA,confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis,M. bovis BCG and M. tuberculosis

The inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre--lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species.     

Identifying control mechanisms of granuloma formation during M. tuberculosis infection using an agent-based model

Infection with Mycobacterium tuberculosis is a major world health problem. An estimated 2 billion people are presently infected and the disease causes approximately 3 million deaths per year. After bacteria are inhaled into the lung, a complex immune response is triggered leading to the formation of multicellular structures termed granulomas. It is believed that the collection of host granulomas either contain bacteria resulting in a latent infection or are unable to do so, leading to active disease. Thus, understanding granuloma formation and function is essential for improving both diagnosis and treatment of tuberculosis. Granuloma formation is a complex spatio-temporal system involving interactions of bacteria, specific immune cells, including macrophages, CD4+ and CD8+ T cells, as well as immune effectors such as chemokine and cytokines. To study this complex dynamical system we have developed an agent-based model of granuloma formation in the lung. This model combines continuous representations of chemokines with discrete agent representations of macrophages and T cells in a cellular automata-like environment. Our results indicate that key host elements involved in granuloma formation are chemokine diffusion, prevention of macrophage overcrowding within the granuloma, arrival time, location and number of T cells within the granuloma, and an overall host ability to activate macrophages. Interestingly, a key bacterial factor is its intracellular growth rate, whereby slow growth actually facilitates survival.     

Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis

The live tuberculosis vaccines Mycobacterium bovis BCG (bacille Calmette-Guérin) and Mycobacterium microti both lack the potent, secreted T-cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein). This is a result of independent deletions in the region of deletion-1 (RD1) locus, which is intact in virulent members of the Mycobacterium tuberculosis complex. To increase their immunogenicity and protective capacity, we complemented both vaccines with different constructs containing the esxA and esxB genes, which encode ESAT-6 and CFP-10 respectively, as well as a variable number of flanking genes. Only reintroduction of the complete locus, comprising at least 11 genes, led to full secretion of the antigens and resulted in specific ESAT-6-dependent immune responses; this suggests that the flanking genes encode a secretory apparatus. Mice and guinea pigs vaccinated with the recombinant strain BCG::RD1-2F9 were better protected against challenge with M. tuberculosis, showing less severe pathology and reduced dissemination of the pathogen, as compared with control animals immunized with BCG alone.     

Genome sequence of Mycobacterium bovis BCG Moreau, the Brazilian vaccine strain against tuberculosis

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here we report the complete genome sequence of M. bovis BCG Moreau, the strain in continuous use in Brazil for vaccine production since the 1920s.     

Protective efficacy against group B streptococcal infection in neonatal mice delivered from preimmunized pregnants

Neonatal mice delivered from mothers preimmunized with heated or formalinized whole cell vaccines of type Ia, Ia/c and III/c group B streptococci were infected with each type of bacteria, and then serum antibodies of mothers and neonates who survived the experiments were measured by enzyme-linked immunosorbent assay. The relationship between the protectivity in neonate mice and the antibody titers to the type specific polysaccharide antigens and the protein c antigen of their sera were examined. In the Ia-immunized group which showed high protection against the type Ia infection, anti-Ia IgG antibody titers were low, and anti-protein c IgG antibody was not detected. Type Ia/c and III/c vaccines were highly effective against both type Ia/c and III/c infection, but less effective in type Ia infection. The protein c antigen was identified in both type strains by the double diffusion assay, and the IgG antibodies to the protein c were significantly high in sera of both maternal mice immunized with types Ia/c or III/c organisms and their newborn infants. High titers of the protein c IgG antibody retained 3 to 4 weeks after the last injection of vaccines which corresponded to the period of pregnancy and lactation. Small amounts of IgM antibody to all antigens were detected only in maternal sera. These results suggest that IgG antibodies to the protein c antigen and to the type-specific polysaccharide antigens are equally important protective factors which are transferable from preimmunized mothers to their newborn infants through placenta and/or lactation.