ISOLATION OF SECRETED MOLECULES OF BCG AN D MYCOBACTERIUM-TUBERCULOSIS IMMUNODOMINANT TARGETS AFTER IMMUNIZATION WITH LIVING BACTERIA

Effective protection against a virulent challenge with Mycobacterium t uberculosis is induced mainly by a previous immunization with a living attenuated related strain. Only a transient and marginal protection i s induced by immunization with bacterial extracts, dead bacteria or av irulent living bacteria. Living and killed bacteria share a number of common antigens. To identify and to purify molecules that are dominant during immunization with living bacteria, a two-step selection proced ure was used. Two groups of guinea-pigs were immunized either with liv ing or with heat-killed BCG. A first group of molecules was selected a ccording to their capacity to elicit delayed-type hypersensitivity (DT H) reactions in guinea-pigs immunized with living BCG and no reaction in guinea-pigs immunized with killed BCG. A second group of molecules was similarly selected on its capacity to interact mainly with antibod ies present in sera of guinea-pigs immunized with living bacteria. Eac h major fraction eluted from a series of high-pressure Liquid chromato graphy columns (gel filtration, DEAE and reverse-phase chromatography) was assayed on its potency to elicit DTH reactions or, after SDS gel electrophoresis and transfer on PVDF sheets, on its capacity to bind a ntibodies. Two groups of molecules were characterized. First, a protei n with an unusual amino-acid composition (40% proline, 12% threonine) was isolated on its potency to elicit DTH reactions on guinea-pigs imm unized with living bacteria. Second, a complex of two molecules specie s was identified and purified on its capacity to interact only with an tibodies present in sera of guinea-pigs immunized with living bacteria , and not at all with antibodies raised after immunization with dead b acteria. The corresponding gene had been cloned and sequenced, it was referred to as apa because of the high percentage of proline (21.7%) a nd alanine (19%) of proteins. These two characterizations of molecules with a high percentage in proline (40% or 20%) prompted us to analyze the role of the proteins for the bacteria and their effects on the im mune response during M. tuberculosis infection.