| Tuberculosis is a major chronic disease that is harmful to human and animals’health. In recent years the emergence of multidrug-resistant Mycobacteriumtuberculosis, and mixed infections with the viruses, makes the treatment oftuberculosis more and more difficult. Therefore, it is urgent to develop safe andefficacious vaccines against tuberculosis. However, M. tuberculosis associatedprotective antigens are poorly identified. Study of hypothetic proteins whosefunctions have not been described yet are depending on the forecast ofbioinformatics. M. tuberculosis proteins Rv3802c, Rv0394c (SEC), Rv0199,E6c10(ESAT6and CFP10fusion protein) are structural proteins of the bacterialcell wall, hyaluronidase, small molecular secretion proteins in early infectingperiod respectively. Previous studies have found that these proteins not onlyshowing a close correlation between the virulence and cellular invasion of M.tuberculosis but also could they be used as drug targets for the screening ordesigning of anti-TB drugs. In addition, since these proteins have goodimmunogenicity, they might be useful in immune protection against tuberculosiswhen applied as recombinant subunit vaccines.Recombinant subunit vaccines by genetic engineering methods need to becomplemented with adjuvant to stimulate a strong immune response and thus togain a good immunity. Salmonella bacteria shed (ghost) is a kind of cell shedswhich doesn’t contain nucleic acid and proteins, but still retains the bacterialsurface antigens of native conformation and biological activities. It was suggestedthat the ghost may also have the adjuvant effects when used as a carrier or vaccine.By now the estimation of applying ghost to the bacterial protective antigens toimprove the immune adjuvant effect has not been reported yet.Our experiments use the four kinds of recombinant proteins of M.tuberculosis, Rv3802c, Rv0394c (SEC), Rv0199and E6C10, which our laboratoryhas already prepared well. We mix them with poultry Salmonellaenteritidis bacteria shed(ghost) and incomplete Freund’s adjuvant(FIA) respectively, and then immunize the SPF BALB/c mice subcutaneously with themixtures. On the14th day after the first immunization and the7th day after thesecond immunization, we detect the antibody and immunoglobulin subclass in themice against the four proteins, and detect IFN-γ and IL-4releasing levels andanalysis the dynamic changes of CD8+T and CD4+T cells of the immunizedmouse spleen lymphocytes after stimulations of four different proteins, usingELISA and flow cytometry methods respectively. We observed the injuries andinflammatory reactions in the injection sites and the damage in the immunizedmouse organs which might be induced by the four proteins throughhistopathological analysis. Moreover we also identify the persistence of antibodyin the mice within one month after the second immunization.The results showed that the ghost and FIA displayed little difference in theadjuvant effect depending on the proteins with higher molecular weight proteins(Rv3802c, Rv0199, SEC). However, the ghost adjuvant could induce higherantibody production than FIA with the lower molecular weight protein (E6c10). Atthe same time all of the four proteins could stimulate the mice to producepersistent antibodies. Through the histopathological observation, we found that allthe experimental groups of mice didn’t undergo obvious trauma, immune adversereactions and inflammatory cell infiltration. It indicated that the bacterial shed(ghost) would have broad application prospects using as a good adjuvant in thearea of subunit vaccines of M. tuberculosis. It also provides a novel idea ofdeveloping multivalent combined vaccines with the ghost’s characteristics of goodadjuvant and lower toxic effects. |
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