Failure of Mycobacterium bovis BCG vaccine: Some Species of

Environmental Mycobacteria Block Multiplication of BCG and Induction of Protective Immunity to Tuberculosis.

Lise Brandt, Joana Feino, Anja Weinreich Olsen, Ben Chilima, Penny Hirsh, Rui Appelberg and Peter

Andersen.

JITENDRA SHANDILYA

The only vaccine available is BCG.

Many different hypothesis have been suggested to explain variation. Such as- s Difference in strain of BCG, Age of vaccination or Methodological differences are the factors for variations.

According to the most widely accepted hypothesis the efficacy of BCG is related with geographic locations, consistently low efficacy in tropical regions, where exposure to nontuberculous bacteria is common.

Neonatal vaccination with BCG imparts protection against childhood manifestation of pulmonary TB., but the efficacy wanes over a period of 10-15 years and so it donot protect against infection of TB in later stage of adult population.

There is convincing evidence by animal experiments that cross protection can partialy mask the efficacy of BCG vaccination.

This study suggest that prior sensitization with env. Mycobacteria can inhibit the multiplication of BCG and thereby prevent the induction of an efficient BCG mediated immune response and protection against TB.

Animal

Pathogen free 6-12 week old CBA/J and C57BL/6J mice.

Bacteria M. Avium(ATCC 15769), M. Safroculaceum (ATCC 19275), M. Vacce (ATCC

15483) are grown on 7H9 broth And M. Tuberculosis on L-J medium until mid log phage of bacterial growth.

Preparation for immunization of mice- Frozen aliquots of bacterial strains were thawed and sonicated for 5 min and viability of each strain was enumerated on 7H11 plate.

M.fortuitum(S78/2) and M. fortuitum(S160/5) are soil isolate from north and south of Karonga and Malawi. These samples are decontaminated with 4% NaOH and cultured at 37o C on nutrient agar based medium to isolate organism from soil.

M. fortuitum(Sp2001), M. chelonae(Sp2015) and M. avium(Sp1891) and M. avium(Sp2011)

are sputum isolates.

These organism are isolated with acidified L-J medium.

Sensitization with Environmental Mycobacteria:

Mice were immunization s.c. in the back three times at 2 week interval with 2*106

CFU of each of three ATCC strain of environmental Mycobacteria.

To clear the remaining bacteria, after 3 weeks of last incubation of, sensitization was followed by 1 month treatment with rifampin, ethambutol, and clarithromycin.

To assess virulence of strains isolated from Karonga & Malawi, mice were infected with 105 CFU of each env. Mycobacteria in 0.2 ml of PBS by I.V. injection.

At the appropriate time points, mice were killed and organ are taken for bacterial enumeration.

Whole organ was homogenized an 0.04% Tween 80 solution in d/w, serial 10 fold dilution were plated on 7H10 medium at 37o C and the number of CFU were determined.

Vaccination

Single dose of BCG Danish 1331 (5*104 CFU) was injected s.c. at the base of the tail.

For subunit vaccination, mice were immunized s.c. three times at 2 week interval with 10µg(per dose) of each ESAT-6 or the Ag85B-ESAT-6 fusion protein, emulsifies in dioctadecylammonium bromide and monophospryl lipid.

M. tuberculosis infection Animals were infected with approx 100 CFU of MTb per lung by aerosol route.

Mice were sacrificed 6 weeks after infection and bacterial number in the lung and spleen were determined.

The effect of BCG or subunit vaccination was expressed as log10 reduction of bacterial count, compared to that in the unvaccinated controlled mice.

Mycobacterial antigen

For preparation of Mycobacterial Antigen, culture filtrate from culture at 6 week, was taken and antigen was precipitated by Ammonium-sulfate precipitation method.

Than the protein concentration was measured by micro bicinchoninic acid method.

LPS content in this precipitate was below (not not influencing the cellular activity). The fusion protein Ag85B-ESAT-6 was produced.

This fusion protein was kept at -80o C until use.

Lymphocyte cultures

Spleen cells or blood cells are taken. RPMI, Antibiotics, Serum and 2-merceptoethanol are added to the

96 well plate. Added ESAT-6 and/or BCG Antigen and a 72 hours incubation was

given. Culture soup was taken and ELISA is done to detect the IFN-γ. ELISPOT is also done to detect the same thing, if the label is very

minute.

Results

1.The multiplication of BCG is inhibited in mice sensitized with certain environmental Mycobacteria.

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