1. 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

2.  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.

3. 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.

4. 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.

5. 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.

6. 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.

7. Results

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

15. THANKS