IFN-γ and IL-5 whole blood response directed against mycolactone polyketide synthase domains in patients withMycobacterium ulceransinfection

Abstract

BackgroundBuruli ulcer is a disease of the skin and soft tissues caused by infection with a slow growing pathogen, Mycobacterium ulcerans. A vaccine for this disease is not available but M. ulcerans possesses a giant plasmid pMUM001 that harbours the polyketide synthase (PKS) genes encoding a multi-enzyme complex needed for the production of its unique lipid toxin called mycolactone, which is central to the pathogenesis of Buruli ulcer. We have studied the immunogenicity of enzymatic domains in humans with M. ulcerans disease, their contacts, as well as non-endemic areas controls.MethodsBetween March 2013 and August 2015, heparinized whole blood was obtained from patients confirmed with Buruli ulcer. The blood samples were diluted 1 in 10 in Roswell Park Memorial Institute (RPMI) medium and incubated for 5 days with recombinant mycolactone PKS domains and mycolyltransferase antigen 85A (Ag85A). Blood samples were obtained before and at completion of antibiotic treatment for 8 weeks and again 8 weeks after completion of treatment. Supernatants were assayed for interferon-γ (IFN-γ) and interleukin-5 (IL-5) by enzyme-linked immunosorbent assay. Responses were compared with those of contacts and non-endemic controls.ResultsMore than 80% of patients and contacts from endemic areas produced IFN-γ in response to all the antigens except acyl carrier protein type 3 (ACP3) to which only 47% of active Buruli ulcer cases and 71% of contacts responded. The highest proportion of responders in cases and contacts was to load module ketosynthase domain (Ksalt) (100%) and enoylreductase (100%). Lower IL-5 responses were induced in a smaller proportion of patients ranging from 54% after ketoreductase type B stimulation to only 21% with ketosynthase type C (KS C). Among endemic area contacts, the, highest proportion was 73% responding to KS C and the lowest was 40% responding to acyltransferase with acetate specificity type 2. Contacts of Buruli ulcer patients produced significantly higher IFN-γ and IL-5 responses compared with those of patients to PKS domain antigens and to mycolyltransferase Ag85A of M. ulcerans. There was low or no response to all the antigens in non-endemic areas controls. IFN-γ and IL-5 responses of patients improved after treatment when compared to baseline results.DiscussionThe major response to PKS antigen stimulation was IFN-γ and the strongest responses were observed in healthy contacts of patients living in areas endemic for Buruli ulcer. Patients elicited lower responses than healthy contacts, possibly due to the immunosuppressive effect of mycolactone, but the responses were enhanced after antibiotic treatment. A vaccine made up of the most immunogenic PKS domains combined with the mycolyltransferase Ag85A warrants further investigation.

Cell-mediated and serology-based tests for Mycobacterium ulcerans disease: A systematic review and meta-analysis

“…Twelve original research studies met the inclusion criteria ( Fig 1 and Table 1), with the earliest from 1952 [25] and the most recent from a WHO conference abstract in 2019 presented by Avumegah [8], which was also Table 1 has been adapted from PhD thesis available online [26]. List of abbreviations not in text: Acyl carrier protein 2& 3(ACP2, ACP3); Acyltransferase with acetate specificity type 1 &2 (ATac1, ATac2); Acyltransferase with propionate specificity (ATp); Enoylreductase (ER); Ketoreductase type A and B (KRA, KR B); Ketosynthase type C (KS C); Ketosynthase domain (Ksalt), dehydratase (DH), and Phorbol 1-myristate 1-acetate (PMA) [34]. Cluster of differentiation 4 (CD4); CD 40 Ligand (CD40L); Tumor necrosis factor alpha (TNFα) [32].…”

“…A summary of these 12 studies are presented in Table 1. Nine out of the 12 CMI and/or serological studies identified were conducted in Africa, specifically Benin [14], Cameroon [31], Cote d'Ivoire [29], Zaire (now Democratic Republic of Congo, DRC) [27], Ghana [15,19,[30][31][32]34] and Uganda [27]. The remaining three studies, were conducted in Australia [8,26,28].…”

“…This was measured by induration on the skin. Other immunodetection methods used were ELISA [8,14,19,26,30,32,34], Western blot for M. ulcerans antibodies and cytokine specific screening [8,19,26,29,31].…”

“…The CMI study by Nausch et al also reported on the TNFα+CD40L-IFNγ-CD4+ T cell subset for BU diagnosis [32]. In 2018, Loglo et al,used PMA, recombinant ACP2, ACP3, Atac1, Atac2, ATp, ER, KR A, KR B, KS C, Ksalt, DH, Ag85Aulc antigens as stimulant for IFN-γ and IL-5 expression ELISA assays [34].…”

“…Positive reactivity among the BU patients ranged from 70% to 85% when using pathogen-derived preparations, but the range of reactivity in controls varied greatly from 3 to 37%. The worst performing recombinant protein was ACP3, showing 47% reactivity among BU patients [34]. Also the panel of proteins used in the CMI study by Loglo et al [34], could not differentiate BU patients from healthy controls [34], however, the 18 kDa shsp was by far a better test antigen.…”