Bacterial culture and transformation. Escherichia coli DH5α, M. smegmatis LR222 and M. bovis BCG (Danish strain) were cultured and transformed, as described earlier [15–17].
DNA manipulations. All restriction endonucleases and the DNA-modifying enzymes such as T4 DNA ligase, Klenow DNA polymerase and T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA) were used according to the recommended protocols. All polymerase chain reaction (PCR) amplifications were carried out using Pfu DNA polymerase (Stratagene GmbH, La Jolla, CA, USA) according to the manufacturer's recommendations.
Construction of the expression vector pSD5.19N. The 19 kDa gene along with its ribosomal binding site and promoter region was PCR amplified using the plasmid pSMT319, which carries the 19 kDa gene along with its native expression signals, as a template. Amplification was carried out by using gene-specific primers 19pUP (GAT CGG CGT CGT CGA AAT C) and 19p3 (TTA GGA ACA GGT CAC CTC G). The blunt-ended PCR product was cloned in EcoRV-digested pSD5 , resulting in pSD5.19N.
Analysis of expression of the 19 kDa antigen in recombinant M. bovis BCG. For analysis of expression, pSD5.19N was electroporated into M. bovis BCG, as described before [15–17]. Transformants were selected on Middlebrook 7H10 medium supplemented with 0.5% glycerol, 1× OADC Middlebrook enrichment and 25 µg/ml kanamycin. The M. bovis BCG transformants were grown in Middlebrook 7H9 medium supplemented with 1× ADC enrichment, 0.5% glycerol, 0.2% Tween-80 and 25 µg/ml kanamycin. Expression of the 19 kDa antigen was monitored both in the cell-free extracts and in the culture supernatants by immunoblot analysis using the monoclonal antibody TB23 against 19 kDa lipoprotein . Thereafter, the membrane was washed thrice with PBS supplemented with 0.05% Tween-20 (PBST) and then incubated for 1 h with HRP-conjugated goat anti-rabbit immunoglobulin G (IgG; 1 : 2500; Jackson Immuno Research Laboratories, Westgrove, PA, USA). The membrane was then washed thrice with PBST and the expression was analysed by using diaminobenzidine and H2O2. Extent of overexpression of the 19 kDa antigen in rBCG19N was measured by subjecting the blots to densitometric analysis by using the NIH image software version 1.52 (NIH image by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Expression levels in the wildtype BCG strain were used as the basal values for comparisons.
Preparation of antigens.M Wild type BCG and recombinant BCG (rBCG) cultures were grown in 7H9 broth, as described earlier [15–17]. Bacterial cells were harvested by centrifugation at 4000 × g for 10 min and antigens were prepared for immunization, as described earlier . Before immunization, the bacterial colony-forming units (CFU) were determined by plating 10-fold serial dilutions of a mildly sonicated cell suspension on Middlebrook 7H10 plates containing kanamycin (25 µg/ml) and 1× OADC.
Recall antigens used in the study. For its use as a recall antigen, the 19 kDa antigen was purified as a His-Tag fusion protein from recombinant E. coli cultures by Ni2+-NTA metal-affinity chromatography . For preparation of the BCG sonicate, wtBCG was grown in 7H9 medium as described above, to an A600 of 2.0–3.0, harvested and sonicated at 4 °C . The sonicate was then centrifuged at 8000 × g for 15 min and proteins in the soluble fraction devoid of cells and cellular debris were used at various concentrations as a recall antigen.
Animals and immunization protocols. Pathogen-free BALB/c mice (6–8 weeks old) obtained from National Center for Laboratory Animals Studies (NCLAS), Hyderabad, were used in the study. Mice in groups of six were immunized intravenously with live 106 CFU of either rBCG19N or wtBCG. The immune responses elicited by wtBCG or rBCG19N on immunization of mice were compared at 4, 8 and 12 weeks post immunization.
Antigen-specific cytokine assays. Proliferation assays were set up using splenocytes prepared from spleens pooled from mice belonging to the same group . Splenocytes were stimulated with either purified 19 kDa antigen or crude BCG sonicate at concentrations ranging from 0.03 µg/ml to 3 mg/ml. Supernatants were harvested after 72 h of culture for the measurement of antigen-specific interferon-gamma (IFN-γ), IL-10 and IL-13 levels using cytokine-specific kits (R&D Systems, Minneapolis, MN, USA). Purified mouse IFN-γ, IL-10 and IL-13 at concentrations ranging from 0.025 to 1 ng/ml were used for generating a standard curve.
Antibody isotyping analysis. Sera from mice belonging to the same group was pooled and analysed for antigen-specific reactivity by enzyme-linked immunosorbent assay. The antibody responses were measured against purified 19 kDa antigen as well as against crude BCG sonicate (500 ng/well). The levels of 19 kDa antigen-specific and BCG sonicate-specific IgG1 and IgG2a antibodies in the various groups of mice were also determined in sera obtained at 4, 8 and 12 weeks post immunization by using the mouse antibody isotyping kit (Gibco BRL, Gaithersburg, MD, USA).
Analysis of protective efficacy of BCG strains in guinea pigs. Random bred guinea pigs (Hartley strain) weighing 200–400 g were purchased either from Pasteur Institute, Conoor, India or from NCLAS, Hyderabad, India. The animals were immunized intradermally in three groups of 10 animals each, with either normal saline or live BCG-Danish strain (1 × 106 CFU) or live rBCG19N (1 × 106 CFU). Eight weeks after immunization, five animals from each of the three groups were subcutaneously challenged with 7.5 × 105 CFU (Challenge dose-1) and the remaining five animals from each group were challenged with 5 × 104 CFU (Challenge dose-2) of M. tuberculosis H37Rv.
All the animals were euthanized at 8 weeks post challenge. Postmortem examination of the guinea pigs was carried out immediately after euthanasia. The gross pathological tissue damage at the site of infection, liver, lung and spleen was scored, as described by Mitchison . The spleen from an individual animal was homogenized in 5 ml of sterile water. Tenfold serial dilutions of the spleen homogenate was plated in duplicates on Löwenstein–Jensen slopes and incubated at 37 °C for 4–6 weeks. The CFU obtained per spleen and the mean splenic CFU for animals belonging to the same group was calculated.
For histopathological analysis, lungs of the animals were also collected and stored in 10% formalin. Portions of these organs (2 cm × 2 cm) were processed for histopathological analysis. The sections were stained with haematoxylin and eosin and the extent of granuloma and type of cellular infiltration in the tissue were microscopically assessed, as described previously [22, 23].
Statistical analysis. All statistical analyses of the data were performed by using the Student's unpaired t-test. A P-value of <0.05 was considered as statistically significant.