T and B immunogenicity of SA5K a secretion antigen of Mycobacterium tuberculosis, in healthy subjects and in tuberculosis patients

Objectives: We recently identified and cloned a novel 8.3 kDa secretory antigen of M. tuberculosis (MTB) and M. bovis bacillus Calmette Guerin (BCG) called SA5K. In the present study, recombinant SA5K containing a histidine hexamer (rSA5K) was expressed in E. coli, purified, and utilized to establish whether it was immunogenic for healthy BCC-vaccinated and nonvaccinated human donors and for tuberculosis (TB) patients. Methods: Enriched T-cell populations were obtained by passage of PBMC through a nylon wool column and stimulated in vitro with BCG culture filtrates (CF), rSA5K, Neg-SA5K (negative control) and PHA. Cultures without stimulus were set up as negative control for cell reactivity. Proliferative responses were evaluated by flow cytometric determination of bromodeoxpridine (BdU) positive cells in the well after 6 days of in vitro stimulation. Recognition of rSASK by human serum antibodies [Ab) was investigated in Western blot experiments with sera obtained from 22 TB patients and 27 healthy donors. Results: A statistically significant difference was observed between healthy vaccinated and nonvaccinated donors in response to rSA5K (P=0.02) as well as to CF (P=0.03). Analysis of the cell populations responding to rSA5K revealed a predominant proliferation of CD4+ T cell subset. Out of 7 TB patients tested only 1 was responding well to rSA5K with a proliferation value more than 5 times the corresponding value for Neg-SA5K. Recognition of rSA5K by serum Ah was generally low in all subject categories. However, at relatively low serum dilution (1 :20), 10/13 sera from healthy BCG-vaccinated subjects and 7/ 13 sera from nonvaccinated subjects (at least 2 of them were PPD+) exhibited a positive recognition of rSA5K. 12/22 sera from TB patients also recognized rSA5K to certain extent. Reactivity of serum Ab to rSA5K was investigated in 11 TB patients over the course of therapy. All of the patients which were positive reactors at the time of the first sampling (3/11) tumed into negative during the course of therapy. Conclusions: While T and B immunogenic potential of rSA5K is generally low in patients with pulmonary TB, vaccination with BCG seems to elicit significant T cell responses and, in a certain extent, B cell responses to rSA5K. Ab titers in TB patients’ sera drop during the course of therapy suggesting that production of SA5K by live bacilli is a necessary stimulus for the maintenance of detectable titers of serum Ab specific for SA5K.