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

[摘要] ENGLISH ABSTRACT: SettingThis study was conducted in the Tygerberg area of Cape Town in South Africa.BackgroundA third of the world's population is latently infected with Mycobacterium tuberculosis,and correlates of protection against progression to active disease urgently need to beidentified to facilitate the development of an effective vaccine against the disease. Theproduction 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 inprotective immunity. The aims of this project were three-fold: (i) to identify promisingTB vaccine candidates by screening a panel of novel MTB antigens, by stimulating wholeblood cultures in vitro with the novel proteins and quantifying the level of IFN-γproduction, (ii) to identify other cytokines and chemokines that may be immunecorrelates of protection using the Luminex fluorescent bead-based technique and (iii) tocompare the performance of the two techniques.MethodsAntigen Screening studyWhole blood of 57 adult and adolescent participants defined as latently infectedindividuals was stimulated with a panel of 78 novel TB-specific, DosR- or RD1-encodedantigens. The 7-day culture supernatants were used in IFN-γ ELISA to quantify the levelof IFN-γ production. Luminex Assay studyWhole blood culture supernatants of 15 HIV negative, TST positive adults were used inthe Luminex LINCO 21-plex cytokine assay. This was done to determine which of 21cytokines, that may be LTBI-associated cytokines, were produced after stimulation with 9TB-specific recombinant antigens, and to quantify their level of expression.ResultsIn the antigen screening study, it was found the majority of the 78 proteins tested wereable 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 andTesatCFP10 (80 – 85% of responders). Ten latency antigens elicited an IFN-γ response in19 – 45% of participants, and five reactivation antigens stimulated a positive reaction in15 – 48% of responders. The category of antigens that elicited the most frequent andhighest responses overall was the resuscitation-promoting factors (Rpf). Over 30% ofparticipants responded to all 5 Rpfs, and the level of responses were equally divided inthe 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 expressionof most cytokines. In addition latency antigens Rv1733c, Rv0569 and Rv2029c alsoinduced moderate-to-high level cytokine expression. A Th1-biased cytokine profile wasobserved, with the preferential expression of pro-inflammatory and cell-mediatedcytokines like IFN-γ, TNF-α, IP-10, MIP1-α and G-CSF being produced. Th2 cytokinesIL-4, IL-5, IL-13 and eotaxin were very poorly expressed or were not expressed atdetectable 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 antigenswith bacterial endotoxins.ConclusionIn this study of latently infected individuals, the pattern of response observed for bothassays is largely a Th1-biased expression profile. The whole blood ELISA method is awell-established assay for quantifying IFN-γ in culture supernatants, and has proven to beeffective here. This study has demonstrated, in humans with LTBI, immune recognitionof these novel MTB-specific antigens as illustrated by the positive IFN-γ levels inducedafter stimulation. The multiplex technology is also a very versatile and sensitive assay,capable of detecting multiple analytes simultaneously in one sample. The multiplex hasbeen valuable here in identifying some antigens as potential vaccine candidates, and asubset of cytokines as potential immune mediators and prognostic indicators in TBinfection.

[发布机构] Stellenbosch University