Identification of immune correlates of natural protection against tuberculosis in a population with a high incidence of latent infection

Golakai, Hawa Jande (2008-03)

Thesis (MScMed)--Stellenbosch University, 2008.

Thesis

ENGLISH ABSTRACT: Setting This study was conducted in the Tygerberg area of Cape Town in South Africa. Background A third of the world’s population is latently infected with Mycobacterium tuberculosis, and correlates of protection against progression to active disease urgently need to be identified to facilitate the development of an effective vaccine against the disease. The production of IFN-γ is recognised as an immune correlate of protection from tuberculosis, but other immune regulators have been implicated in playing a significant role in protective immunity. The aims of this project were three-fold: (i) to identify promising TB vaccine candidates by screening a panel of novel MTB antigens, by stimulating whole blood cultures in vitro with the novel proteins and quantifying the level of IFN-γ production, (ii) to identify other cytokines and chemokines that may be immune correlates of protection using the Luminex fluorescent bead-based technique and (iii) to compare the performance of the two techniques. Methods Antigen Screening study Whole blood of 57 adult and adolescent participants defined as latently infected individuals was stimulated with a panel of 78 novel TB-specific, DosR- or RD1-encoded antigens. The 7-day culture supernatants were used in IFN-γ ELISA to quantify the level of IFN-γ production. Luminex Assay study Whole blood culture supernatants of 15 HIV negative, TST positive adults were used in the Luminex LINCO 21-plex cytokine assay. This was done to determine which of 21 cytokines, that may be LTBI-associated cytokines, were produced after stimulation with 9 TB-specific recombinant antigens, and to quantify their level of expression. Results In the antigen screening study, it was found the majority of the 78 proteins tested were able to induce a positive IFN-γ response. The classic TB antigens were used as controls, and the frequency of responses was highest after stimulation with ESAT-6 and TesatCFP10 (80 – 85% of responders). Ten latency antigens elicited an IFN-γ response in 19 – 45% of participants, and five reactivation antigens stimulated a positive reaction in 15 – 48% of responders. The category of antigens that elicited the most frequent and highest responses overall was the resuscitation-promoting factors (Rpf). Over 30% of participants responded to all 5 Rpfs, and the level of responses were equally divided in the low and moderate-to-high levels, with an additional 5% of responses in the high (>1000pg/ml) range. In the Luminex study, the positive stimulant TesatCFP10 consistently induced expression of most cytokines. In addition latency antigens Rv1733c, Rv0569 and Rv2029c also induced moderate-to-high level cytokine expression. A Th1-biased cytokine profile was observed, with the preferential expression of pro-inflammatory and cell-mediated cytokines like IFN-γ, TNF-α, IP-10, MIP1-α and G-CSF being produced. Th2 cytokines IL-4, IL-5, IL-13 and eotaxin were very poorly expressed or were not expressed at detectable levels. A very strong induction of IL-6, IL-8 and MCP-1 was observed, but this cytokine/chemokine association suggested contamination of the recombinant antigens with bacterial endotoxins. Conclusion In this study of latently infected individuals, the pattern of response observed for both assays is largely a Th1-biased expression profile. The whole blood ELISA method is a well-established assay for quantifying IFN-γ in culture supernatants, and has proven to be effective here. This study has demonstrated, in humans with LTBI, immune recognition of these novel MTB-specific antigens as illustrated by the positive IFN-γ levels induced after stimulation. The multiplex technology is also a very versatile and sensitive assay, capable of detecting multiple analytes simultaneously in one sample. The multiplex has been valuable here in identifying some antigens as potential vaccine candidates, and a subset of cytokines as potential immune mediators and prognostic indicators in TB infection.

AFRIKAANSE OPSOMMING: Studie-area Hierdie studie was gedoen in die Tygerberg area van Kaapstad in Suid-Afrika. Agtergrond ‘n Derde van die wêreld se bevolking is latent geïnfekteer met Mycobacterium tuberculosis en korrelate van beskerming teen die siekte moet geïdentifiseer word om die ontwikkeling van ‘n effektiewe enstof te fasiliteer. Die produksie van IFN-γ is welbekend as ‘n immuunkorrelaat van beskerming teen tuberkulose (TB), maar ander immuunreguleerders speel ook ‘n belangrike rol in beskermende immuniteit. Die doelwitte van hierdie projek was drievoudig: (i) om belowende TB-entstof kandidate te identifiseer deur die sifting van ‘n paneel van nuwe MTB antigene mbv die in vitro stimulasie van volbloed kulture, ii) om ander sitokiene en chemokiene as immuunkorrelate van beskerming te identifiseer deur van die Luminex fluorescent bead-based tegniek gebruik te maak, en (iii) om die twee tegnieke te vergelyk op grond van hul prestasie as prognostiese of siftings metodes in latente infeksie. Metodes Antigeen siftings studie Volbloed van 57 volwasse en adolessente deelnemers, geïdentifiseer as latent geïnfekteerde individue, was gestimuleer met ‘n paneel van 78 nuwe TB-spesifieke DosR- or R-gekodeerde antigene. Die 7-dae kultuur supernatante was gebruik in ‘n IFN-γ ELISA om die hoeveelheid IFN-γ produksie the kwantifiseer. Luminex assay studie Volbloed kultuur supernatante van 15 HIV negatiewe, TST positiewe volwassenes was gebruik in die Luminex LINCO 21-plex cytokine assay. Dit was gedoen om die tipes en hoeveelheid ander LTBI-geassosieerde sitokienes te identifiseer wat geproduseer word na stimulasie met 9 TB-spesifieke rekombinante antigene. Resultate In die antigeen siftings studie is gevind dat die meerderheid van die 78 getoetste proteïene ‘n positiewe IFN-γ reaksie kon induseer. Vir die kontroles was die frekwensie van reaksies die hoogste na stimulasie met ESAT-6 en TesatCFP-10 (80 – 85% van reageerders). Tien latensie antigene was gereeld herken deur 19 – 45% van deelnemers en vyf reaktiverings-antigene het ‘n positiewe reaksie in 15 – 48% van reageerders gestimuleer. Die kategorie van antigene wat die meeste en hoogste response veroorsaak het, was die resusitasie-promoterende faktors (Rpf). Meer as 30% van deelnemers het op al 5 Rpfs gereageer en die vlak van reaksies was gelyk verdeel in die lae en matig-tot-hoog vlakke, met ‘n addisionele 5% van reaksies in die hoë (>1000pg/ml) reeks. In die Luminex studie het die positiewe stimulant TesatCFP-10 konsekwent die positiewe uitdrukking van die meeste sitokiene geïnduseer. Saam met dit het die latente antigene Rv1733c, Rv0569 en Rv2029c ook matige-toe-hoë vlakke van sitokien uitdrukking geïnduseer. ‘n Th1-gebaseerde sitokien profiel was waargeneem, met die begunstigde uitdrukking van pro-inflammatoriese en sel-gemedieerde sitokiene soos IFN-γ, TNF-α, IP-10, MIP1-α en G-CSF. Th2 sitokiene IL-4, IL-5, IL- 13 en eotaksien was of baie sleg uitgedruk of onder naspeurbare vlakke uitgedruk. ‘n Baie sterk induksie van IL-6, IL-8 en MCP-1 was waargeneem, maar hierdie sitokiene/chemokiene assosiasie stel moontlik kontaminasie van die rekombinante antigene met bakteriële endotoksiene voor. Samevatting Die reaksiepatroon wat in hierdie studie tussen die twee toetse waargeneem is, was grootliks ‘n Th1-gebaseerde uitdrukkingsprofiel vir latente infeksie met TB. Die volbloed ELISA metode is a betroubare gevestigde toets vir die kwantifisering van IFN-γ in kultuur supernatante, wat ook in hierdie studie bewys is om effektief te wees. Hierdie studie het gedemonstreer dat die nuwe TB-spesifieke antigene effektief positiewe IFN-γ response in mense met LTBI induseer. Die multipleks tegnologie is ook ‘n baie veelsydige en sensitiewe toets, wat in staat is om veelvoudige analite gelyktydig in een monster te kan opspoor. In hierdie studie was dit veral waardevol in die identifisering van ander moontlike antigene as prognostiese kandidate en sitokiene as immuunbemiddelaars in TB-infeksie.

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