Humoral immune responses in mice immunized with region of difference DNA vaccine constructs of pUMVC6 and pUMVC7.

BACKGROUND: We aimed to study the antigen-specific antibody responses in mice immunized with recombinant DNA vaccines constructs of pUMVC6 and pUMVC7, containing RD1 and RD9 genes of Mycobacterium tuberculosis. METHODS: We immunized mice with the parent and recombinant plasmids and sera were collected and tested for antibodies against pure recombinant proteins of RD1 (PE35, PPE68, EsxA, EsxB) and RD9 (EsxV), peptide mixtures of each protein and their individual peptides using enzyme-linked immunosorbent assays. The optical density (OD) values were measured at 405 nm. E/C (OD in antigen-coated wells/OD in antigen uncoated wells) were calculated, and the values of E/C>2 were considered positive. RESULTS: RD1 and RD9 antigen-specific antibodies were detected in sera of mice immunized with the recombinant DNA vaccine constructs (E/C >2.0). With respect to peptide mixtures and single peptides, only PE35mixand P6 of PE35; PPE68mixand P19, P24 of PPE68 showed antibody reactivity with sera of mice immunized with the corresponding recombinant pUMVC6 and/or pUMVC7 DNA vaccine constructs. CONCLUSIONS: The results confirm in vivo expression and immunogenicity of all the five RD1 and RD9 genes cloned in both of the DNA vaccine vectors.

Pharmaceutical aerosols for the treatment and prevention of tuberculosis.

Historically, pharmaceutical aerosols have been employed for the treatment of obstructive airway diseases, such as asthma and chronic obstructive pulmonary disease, but in the past decades their use has been expanded to treat lung infections associated with cystic fibrosis and other respiratory diseases. Tuberculosis (TB) is acquired after inhalation of aerosol droplets containing the bacilli from the cough of infected individuals. Even though TB affects other organs, the lungs are the primary site of infection, which makes the pulmonary route an ideal alternative route to administer vaccines or drug treatments. Optimization of formulations and delivery systems for anti-TB vaccines and drugs, as well as the proper selection of the animal model to evaluate those is of paramount importance if novel vaccines or drug treatments are to be successful. Pharmaceutical aerosols for patient use are generated from metered dose inhalers, nebulizers, and dry powder inhalers (DPIs). In addition to the advantages of providing more efficient delivery of the drug, low cost, and portability, pharmaceutical dry powder aerosols are more stable than inhalable liquid dosage forms and do not require refrigeration. Methods to manufacture dry powders in respirable sizes include micronization, spray drying, and other proprietary technologies. Inhalable dry powders are characterized in terms of their drug content, particle size, and dispersibility to ensure deposition in the appropriate lung region and effective aerosolization from the device. These methods will be illustrated as they were applied for the manufacture and characterization of powders containing anti-tubercular agents and vaccines for pulmonary administration. The influence of formulation, selection of animal model, method of aerosol generation, and administration on the efficacy demonstrated in a given study will be illustrated by the evaluation of pharmaceutical aerosols of anti-TB drugs and vaccines in guinea pigs by our group.

Whole blood assays to identify Th1 cell antigens and peptides encoded by Mycobacterium tuberculosis-specific RD1 genes.

OBJECTIVE: To identify Th1 cell-stimulating antigens/peptides encoded by the genes predicted in the Mycobacterium tuberculosis-specific genomic region of difference (RD)1, deleted in Mycobacterium bovis Bacille Calmette-Guérin(BCG), by using synthetic peptides and whole blood from tuberculosis (TB) patients. MATERIALS AND METHODS: Heparinized peripheral blood was obtained from culture-proven pulmonary TB patients (n = 16) attending the Chest Disease Hospital, Kuwait. Whole blood was diluted with tissue culture medium RPMI-1640 and tested for Th1 cell stimulation using antigen-induced proliferation and interferon-gamma (IFN-gamma) secretion assays. The antigens included a peptide pool of 220 peptides covering the sequence of 12 open reading frames (ORFs) of RD1 (RD1(mix)), peptide pools of RD1 ORF5 (ORF5(mix)), ORF6 (ORF6(mix)) and ORF7 (ORF7(mix)), and individual peptides of ORF6 (P6.1-P6.6) and ORF7 (P7.1-P7.6). M. tuberculosis culture filtrate, cell walls and whole-cell M. bovis BCG were used as complex mycobacterial antigens. The results obtained with different antigens and peptides were statistically analyzed for significant differences using Z test. RESULTS: The complex mycobacterial antigens (culture filtrate, cell walls and M.bovis BCG) and RD1(mix) induced comparable (p > 0.05) positive antigen-induced proliferation and IFN-gamma responses with whole blood from TB patients. However, the positive IFN-gamma responses induced by ORF6(mix) and ORF7(mix) were higher than ORF5(mix). Among the individual peptides, P6.4 and P7.1 of ORF6 and ORF7, respectively, induced the highest IFN-gamma responses, suggesting that these peptides represented the immunodominant Th1 cell epitopes of RD1 ORF6 and ORF7 in the patients tested. CONCLUSION: The whole blood assays with synthetic peptides are useful to identify Th1 cell antigens/peptides encoded by genes located in M. tuberculosis-specific genomic regions.