B-Cell Epitopes in NTS-DBL1 alpha of PfEMP1 Recognized by Human Antibodies in Rosetting Plasmodium falciparum

Plasmodium falciparum is the most lethal of the human malaria parasites. the virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1 alpha-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1 alpha of IT4(var60)) expressed by parasite FCR3S1.2. the plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1 alpha domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. the other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. in conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1 alpha of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. the data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.Swedish Research Council (VR)Swedish Academy of Sciences (KVA, Soderberg Foundation)Karolinska Institutet-DPAEU Network of Excellence EviMalarKarolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Stockholm, SwedenKarolinska Inst, Dept Lab Med, Therapeut Immunol TIM, Stockholm, SwedenKarolinska Univ Hosp, CAST, Huddinge, SwedenUniv Estadual Campinas, Dept Biochem, Campinas, SP, BrazilWeb of Scienc

Antileishmanial Activity of the Terpene Nerolidol

The activity of nerolidol, a sesquiterpene used as a food-flavoring agent and currently under testing as a skin penetration enhancer for the transdermal delivery of therapeutic drugs, was evaluated against Leishmania species. Nerolidol inhibited the growth of Leishmania amazonensis, L. braziliensis, and L. chagasi promastigotes and L. amazonensis amastigotes with in vitro 50% inhibitory concentrations of 85, 74, 75, and 67 μM, respectively. The treatment of L. amazonensis-infected macrophages with 100 μM nerolidol resulted in 95% reduction in infection rates. Inhibition of isoprenoid biosynthesis, as shown by reduced incorporation of [2-(14)C]mevalonic acid (MVA) or [1-(14)C]acetic acid precursors into dolichol, ergosterol, and ubiquinone, was observed in nerolidol-treated promastigotes. This drug effect can be attributed to the blockage of an early step in the mevalonate pathway, since incorporation of the precursor [1(n)-(3)H]farnesyl pyrophosphate in polyisoprenoids is not inhibited by nerolidol. L. amazonensis-infected BALB/c mice were treated with intraperitoneal doses of 100 mg/kg/day for 12 days or topically with 5 or 10% ointments for 4 weeks. Significant reduction of lesion sizes in nerolidol treated mice was observed for both treatment routes. However, long-term follow up indicated that the disease was not cured in this highly susceptible animal model. Nonetheless, the in vitro activity of nerolidol against these parasites may prove a useful tool for the development of new drugs for the treatment of leishmaniasis. In addition, biosynthesis of dolichols with 11 and 12 isoprene units was identified in Leishmania, as described for other trypanosomatids and Apicomplexa

Peptides targeted by rosette disruptive antibodies in FCR3S1.2 iRBC.

<p>List of peptides, recognized on the peptide array, with a positive correlation with the ability of the plasma sample to disrupt FCR3S1.2 rosettes. Peptides indicated in bold are conserved between different parasite sequences and are further described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113248#pone.0113248.s002" target="_blank">Figure S2</a>.</p><p>Peptides targeted by rosette disruptive antibodies in FCR3S1.2 iRBC.</p

Naturally acquired antibodies to NTS-DBL1α.

<p>A) IgG levels in 134 human plasma samples against the recombinant NTS-DBL1α^It4var60 as detected by ELISA. Non immune indicates 20 non immune Swedish donors. Uncomplicated and severe indicate patients from Cameroon. B) Correlation between patient age and presence of antibodies in the plasma towards the recombinant NTS-DBL1α^It4var60 as measured by ELISA. C) Correlation between ability of the plasma sample to disrupt FCR3S1.2 iRBC rosettes and presence of antibodies towards to NTS-DBL1α^It4var60 as measured by ELISA. D) Correlation between ability of the sample to disrupt FCR3S1.2 iRBC rosettes and patient age. E) Correlation between ability of the plasma sample to recognize FCR3S1.2 iRBC surface as detected by FACS and presence of antibodies towards to NTS-DBL1α^It4var60 as measured by ELISA. F) Correlation between ability of the plasma sample to recognize FCR3S1.2 iRBC surface as detected by FACS and patient age.</p

Reactivity of human plasma to a peptide array of the NTS-DBL1α^It4var60.

<p>A subset of 26 plasma samples (red dots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113248#pone-0113248-g001" target="_blank">figure 1C</a>; further described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113248#pone-0113248-t001" target="_blank">Table 1</a>) were tested on a peptide microarray covering the NTS-DBL1α^It4var60 domain. 9 plasma samples were able to disrupt FCR3S1.2 iRBC rosettes while 17 were not. The mean value for the reactivity index of each sample group was compared to the mean reactivity index of 8 non-immune control samples. Green arrows indicate peptides with differential recognition between the groups.</p

Peptides associated with the ability to disrupt rosettes of the FCR3S1.2 strain.

<p>Reactivity towards five peptides (indicated by green arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113248#pone-0113248-g004" target="_blank">figure 4</a>) was correlated with plasma sample ability to disrupt FCR3S1.2 rosettes. A) Scatter plots showing the correlation between specific peptide recognition and the ability to disrupt rosettes of FCR3S1.2 iRBC. B) Reactivity index measured in peptide array to the same peptides according to their grouping: rosette disruptive (RD), non-rosette disruptive (Non-RD) and Swedish non-immune control samples (NI).</p

Peptide array sample groups.

<p>Summary of the subset of samples further analyzed on the peptide array (shown as red dots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113248#pone-0113248-g001" target="_blank">Figure 1C</a>). The table summarizes the sample name (NR: non rosette disruptive; RD: rosette disruptive), surface reactivity towards FCR3S1.2 iRBC (MFI), the ELISA reactivity towards the NTS-DBL1α domain and ability to disrupt the FCR3S1.2 iRBCs rosettes.</p><p>Peptide array sample groups.</p

Orthogonal Partial Least Square analysis (OPLS) of peptide array data for It4var60.

<p>Distribution of samples on the OPLS model according to their reactivity on the peptide array (top). VIP ranking score for the OPLS analysis (Bottom). Red arrows indicate the peptides correlated with the ability to disrupt rosettes as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113248#pone-0113248-g005" target="_blank">figure 5</a>.</p

Principal Component Analysis of clinical and immunological data.

<p>Data analysis from Cameroonian samples where clinical parameters and immunological data were analyzed using Principal Component Analysis. The principal component analysis generated a model that explains 23% of the data segregation (R2X  = 0.234545). Clinical parameters: temperature (Temp), breath rate (breath), pulse rate (pulse), glucose level (glucose), haemoglobin (Hb), parasitemia, age, rosetting rates (RR, after collection from patients). Immunological parameters: plasma sample recognition of NTS-DBL1α It4var60 domain (ELISA), ability to disrupt FCR3S1.2 rosettes (RD) and ability to recognize FCR3S1.2 iRBC surface by flow cytometry (Surface reactivity).</p