Investigation of Mycobacterium tuberculosis protein expression and analysis of humoral immune responses of TB patients

Pheiffer, Carmen (2004-12)

Thesis (PhD)--Stellenbosch University, 2004.

Thesis

ENGLISH ABSTRACT: New agents for the diagnosis, prevention and treatment of tuberculosis are urgently required. Yet, despite extensive tuberculosis research over recent years, no new drugs, vaccines or diagnostics have been identified to date. It is widely speculated that the major obstacle to the identification of new therapies is the lack of understanding of the hostpathogen interaction. This study has investigated whether patterns of antigen expression correlate with molecular epidemiological data and strain virulence through the analysis of protein expression and antigen recognition profiles of different M tuberculosis clinical isolates. Using polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and Western blotting, protein expression and antigen recognition by two genotypically different clinical strains that differed in their frequency in the study population have been compared. In addition to differences in protein expression and antigen recognition between the clinical strains and the reference strain H37Rv, protein expression differences between the clinical strains themselves were observed which may relate to strain frequency and virulence. Differential protein expression by M tuberculosis strains, may explain the heterogeneous host humoral immune response and why no fully effective serodiagnostic test has been developed to date. To explore this hypothesis, the potential of serodiagnosis in this community, where patients are infected with a wide variety of genotypically distinct strains, was investigated. IgG levels to three mycobacterial antigens showed that serodiagnosis of TB is possible in this community, despite infection by a wide variety of genotypically different M tuberculosis strains. Disease episode affected antibody levels, suggesting that care should be taken when evaluating serological diagnosis for repeat episode patients. This study has shown that M tuberculosis protein expression is dynamic and that the bacillus presents a hypervariabie array of antigens to the host immune system. It is likely that different antigens become immunodominant as antituberculosis chemotherapy progresses, and that these differentially expressed antigens may be tracked as predictors of treatment outcome. This hypothesis was tested by correlating Ag85-specific IgG with treatment response, as assessed by sputum smear conversion after two months of antimycobacterial chemotherapy. No significant correlation between antibody levels and treatment responses was observed, suggesting that antibodies may not be useful surrogate markers or that the incorrect antibody type or mycobacterial antigen were selected. Results were consistent with previous findings where patient-to-patient variation dictated the host humoral response. The results obtained in this study have demonstrated that although bacteriological factors may influence strain prevalence due to antigen variation and immune evasion, both bacteriological and host factors affect humoral immunity. Differential protein expression by M tuberculosis strains has potentially important implications for serodiagnosis and the development of subunit or DNA vaccines, by suggesting that multi-antigen cocktails should be used. Differential protein expression may also explain why patients do not develop adequate protective immunity and are susceptible to reinfection.

AFRIKAANSE OPSOMMING: Daar is 'n dringende behoefte vir nuwe middels vir die diagnosering, voorkoming en behandeling van tuberkulose. Ondanks intense tuberkulose navorsing gedurende die afgelope paar jaar, is daar geen nuwe tuberkulose medikasie, vaksines of diagnostiese metodes geïdentifiseer nie. Daar word gespekuleer dat die hoof struikelblok vir die identifisering van nuwe medikasie die onkunde oor die tuberkulose patogeen is. Deur die analise van proteien-uitdrukking en antigeen-erkenning profiele van verskillende M. tuberculosis kliniese isolate is daar tydens hierdie studie ondersoek ingestel of die patroon van antigeen uitdrukking korreleer met molekulêre epidemiologiese data and stam-virulensie. Proteien-uitdrukking en antigeen-erkenning deur twee genotipies verskillende kliniese stamme wat verskil in hul frekwensie in die bestudeerde populasie, is vergelyk deur middel van poli-akrielamied gel elektroforese, ensiem-gekoppelde immuunabsorberende analise en Westelike oordrag. Addisoneel tot die verskille in proteienuitdrukking en antigeen-ekenning tussen kliniese stamme en die verwysingstam H37Rv, is daar ook verskille aangedui tussen die kliniese stamme self wat kan dui op stam frekwensie en virulensie. Differensiële proteien-uitdrukking deur M. tuberculosis stamme, kan moontlik die heterogene gasheer se humorale immuunreaksie verduidelik en daarmee saam die rede waarom daar nie tot op hede 'n effektiewe sero-diagnostiese toets ontwikkel is nie. Daar is dus ondersoek ingestel na die potensiaal van sero-diagnose in 'n gemeenskap waar pasiënte geïnfekteer is met 'n wye verskeidenheid genotipiese stamme. Die IgG vlakke van drie mikobakteriële antigene het aangedui dat sero-diagnose van tuberkulose moontlik is in hierdie gemeenskap, ten spyte van infektering deur 'n wye verskeidenheid genotipies-verskillende M. tuberculosis stamme. Die tussenspel van die siekte het teenliggaampie-vlakke beïnvloed wat daarop dui dat daar versigtig moet gelet word tydens die evaluering van serologiese diagnose van geïnfekteerde pasiënte wat voorheen siek was. Hierdie studie toon dat M. tuberculosis proteïen-uitdrukking dinamies is en dat die bacillus 'n groot variëteit van antigene tot die immuun sisteem bied. Dit is moontlik dat verskillende antigene immuun dominant kan word soos wat antituberkulose chemoterapie toeneem, en dat hierdie verskillend-uitgedrukte antigene as 'n gevolg daarvan gebruik kan word as voorspellers vir behandeling. Hierdie hipotese is getoets deur die korrelering van Ag85-spesifieke IgG met die reaksie op behandeling soos geëvalueer deur speeksel-monster verandering na twee maande se anti-mikobakteriële chemoterapie. Daar was geen noemenswaardige korrelasie tussen teenliggaampie vlakke en die reaksie op behandeling nie, wat daarop dui dat die teenliggaampies nie toepaslike surrogaat merkers is nie of dat die verkeerde teenliggaampie-tipe of mikobakteriële antigeen geselekteer is. Hierdie resultate bevestig vorige bevindinge waar pasiënt-tot-pasiënt verskille die gasheer se humorale immuunreaksie gedikteer het. Die resultate wat uit hierdie studie volg dui dat alhoewel bakteriologiese faktore die stam-frekwensie kan beïnvloed as gevolg van antigeen-variasie en immuun-ontduiking, kan beide bakteriologiese en gasheer faktore die humorale immuunreaksie beïnvloed. Differensiële proteiën uitdrukking deur 'n verskeidenheid M. tuberculosis stamme het potensieël belangrike toepassings vir sero-diagnose en die ontwikkeling van subeenheid of DNS vaksines wat impliseer dat multi-antigeen mengsels gebruik moet word. Differensiële proteiën uitdrukking mag ook verduidelik waarom pasiënte nie 'n voldoende beskermende immuniteit opbou nie en sodoende ontvanklik is vir her-infeksie.

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