Mycolactone diffuses from Mycobacterium ulcerans-infected tissues and targets mononuclear cells in peripheral blood and lymphoid organs.

Buruli ulcer (BU) is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-gamma responses in BU patients remains unclear.Here we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans-infected mice coincided with alterations in the functions of circulating lymphocytes.In addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease

Mycolactone targets mononuclear cells in blood and spleen cell suspensions.

<p>The distribution of mycolactone in the gradient density fractions of whole blood or spleen cell suspensions is shown. Mycolactone was added to whole blood (20 µg/ml) or spleen cell suspensions (20 µg/spleen) and incubated for 4 h or 1 h, respectively. The amount of mycolactone distributing in each gradient fractions was then determined by ESI-LC-MS quantitative analysis of their acetone-soluble lipid extracts. Data are mean percentages and SD of duplicate measurements, and are representative of two independent experiments.</p

Presence of mycolactone in serum during <i>M. ulcerans</i> infection.

<p><i>A)</i> The dose-dependent immunosuppressive activity of mycolactone on the IL-2 production of Jurkat T cells is shown. IL-2 concentration was measured in culture supernatants of Jurkat T cells incubated with mycolactone A/B for 6 h prior to stimulation with PMA/ionomycin for 16 h. <i>B)</i> The effect of control mouse sera on stimulation-induced production of IL-2 by Jurkat T cells is shown, by comparison to cells incubated in the absence of serum. The graph shows that this inhibitory effect can be neutralized by heating the control mouse sera at 90°C for 10 min (HT), prior to addition onto Jurkat T cells. Controls include cells in the absence of stimulation (NS), and cells activated in the presence of sera spiked with mycolactone (400 ng/ml), then heat-treated. <i>C)</i> Immunosuppressive activity of sera harvested from mice infected with wild-type <i>M. ulcerans</i> (wtMu) or the mycolactone deficient mutant mup045Mu, as compared to sera from uninfected animals. Data are mean and SD of duplicate measurements of IL-2 production by Jurkat T cells activated in the presence of mouse sera (n = 4), and are representative of two independent experiments.</p

<i>M. ulcerans</i>–produced mycolactone diffuses in internal organs of experimentally infected mice.

<p>The distribution of mycolactone is shown in internal organs of mice ten weeks post sc infection with 10⁴<i>M. ulcerans</i> bacilli. The amount of mycolactone was determined by ESI-LC-MS analysis of acetone-soluble lipid extracts prepared from pools of 5 homogenized organs. Data correspond to the calculated amount of total mycolactones (A/B and C forms) per organ, nd: not detected.</p

Pharmacokinetics of mycolactone<i> in vivo.</i>

<p>Panel A shows the kinetics of mycolactone concentration in the circulating blood of mice following an intravenous (IV), intraperitoneal (IP) or subcutaneous (SC) injection of 300 µg of ¹⁴C-labeled mycolactone. Panel B shows radioactivity levels in various internal organs 24 h post injection of mycolactone via these three administration routes. The left axis indicates cpm levels and the right one the corresponding mycolactone concentrations, for a 300 cpm/µg activity. Data were acquired on pools of blood samples or homogenized organs (n = 3). Panel C illustrates the preferential distribution of mycolactone in the spleen, versus kidney and liver, following injection via the IV (n = 5) or the SC (n = 5) route in three independent experiments. Radioactivity levels in each organ were compared with the Friedman Test (Nonparametric Repeated Measures ANOVA) and Dunn's multiple comparison post-test (*: p<0,05; **: p<0,01).</p

Mycolactone is present in the mononuclear cells of <i>M. ulcerans</i>-infected animals.

<p>C57BL/6 mice (n = 3) were infected by footpad injection of 10⁴ wtMu or mup045Mu bacilli. The distribution of mycolactone in PBMCs and in the mononuclear cell fraction of draining lymph nodes (DLNs), inguinal lymph nodes (ILNs) and spleens (Spleen) is shown 6 weeks post infection. Mononuclear cells were isolated from pooled samples of whole blood (1 ml), pooled DLNs (n = 3), or from the ILNs and spleens of 3 individual mice. Acetone-soluble lipid were then extracted from cell pellets and mycolactone concentrations determined by quantitative LC/MS-MS analysis. Means and SD are shown for ILNs and spleens. C) IL-2 production after whole blood stimulation with anti-CD3 and -CD28 antibodies for 24 h. Data are mean and SD of IL-2 concentrations, as measured in duplicate for pooled blood samples (n = 4) after 2, 4 and 6 weeks of infection, and are representative of three independent experiments. Differences in IL-2 concentration between groups were analyzed by one-way ANOVA (*: p<0,05; **: p<0,01).</p

A nonintegrative lentiviral vector-based vaccine provides long-term sterile protection against malaria.

Trials testing the RTS,S candidate malaria vaccine and radiation-attenuated sporozoites (RAS) have shown that protective immunity against malaria can be induced and that an effective vaccine is not out of reach. However, longer-term protection and higher protection rates are required to eradicate malaria from the endemic regions. It implies that there is still a need to explore new vaccine strategies. Lentiviral vectors are very potent at inducing strong immunological memory. However their integrative status challenges their safety profile. Eliminating the integration step obviates the risk of insertional oncogenesis. Providing they confer sterile immunity, nonintegrative lentiviral vectors (NILV) hold promise as mass pediatric vaccine by meeting high safety standards. In this study, we have assessed the protective efficacy of NILV against malaria in a robust pre-clinical model. Mice were immunized with NILV encoding Plasmodium yoelii Circumsporozoite Protein (Py CSP) and challenged with sporozoites one month later. In two independent protective efficacy studies, 50% (37.5-62.5) of the animals were fully protected (p = 0.0072 and p = 0.0008 respectively when compared to naive mice). The remaining mice with detectable parasitized red blood cells exhibited a prolonged patency and reduced parasitemia. Moreover, protection was long-lasting with 42.8% sterile protection six months after the last immunization (p = 0.0042). Post-challenge CD8+ T cells to CSP, in contrast to anti-CSP antibodies, were associated with protection (r = -0.6615 and p = 0.0004 between the frequency of IFN-g secreting specific T cells in spleen and parasitemia). However, while NILV and RAS immunizations elicited comparable immunity to CSP, only RAS conferred 100% of sterile protection. Given that a better protection can be anticipated from a multi-antigen vaccine and an optimized vector design, NILV appear as a promising malaria vaccine

NILV elicit as frequent blood CSP-specific T cells as RAS after 3 injections.

<p>BALB/c mice (n = 6/group) were immunized 3 times by IP injections of NILV. They were primed by administration of NILV particles encoding Py CSP and pseudotyped with VSV-G IND at the dose of 100 or 1500 ng p24/mouse. They were boosted 2 months later with 1500 ng p24 of NILV particles pseudotyped with VSV-G NJ, and boosted again 5 months later with 1500 ng p24 of NILV particles pseudotyped with the glycoprotein from Cocal virus. Additionally, mice (n = 6) from the same batch were immunized 3 times by IV injection with RAS at monthly intervals (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g003" target="_blank"><b>Figure 3A</b></a>). The frequency of S9I-specific blood CD8+ cells was followed over time by S9I/K^d tetramer staining after NILV (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g003" target="_blank"><b>Figure 3B</b></a>) and RAS immunizations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g003" target="_blank"><b>Figure 3C</b></a>). Data from individual mice and means are shown. The Y-axis uses a logarithmic scale.</p

NILV are as immunogenic as ILV when 10-times more particles are injected.

<p>BALB/c mice (n = 5/group) were immunized by IM injection with various doses (expressed as TU/mouse) of lentiviral vector particles encoding Py CSP, either NILV (□) or ILV (▪). Ten days later, specific cellular immune responses were assessed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2A</b></a>). The frequency of S9I-specific blood CD8+ cells was assessed by S9I/K^d tetramer staining, and the frequency of IFNg secreting splenocytes in response to overnight restimulation with S9I or I10L peptides was measured by IFNg elispot assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2B</b></a>). Means + SD are shown. BALB/c mice (n = 3/group) were IM immunized with NILV (□) or ILV (▪) at the dose of 5E+08 or 5E+07 TU/mouse respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2C</b></a>). The frequency of S9I-specific blood CD8+ cells was followed over time by tetramer staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2D</b></a>). At day 24 post-immunization, spleen cellular response was analyzed by S9I/K^d tetramer staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2E</b></a>), by IFNg elispot in response to S9I and I10L peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2F</b></a>), and by intracellular staining of 3 cytokines, IFNg, IL2 and TNFa, in response to S9I (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2G</b></a>). Cells secreting individual (green), 2 (blue) or 3 (red) cytokines are shown. Anti-(QGPGAP)₂-specific IgG at day 21 post-immunization were quantified by ELISA and expressed as titers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048644#pone-0048644-g002" target="_blank"><b>Figure 2H</b></a>). Medians + range are shown.</p