Immune response and biochemistry of calves immunized with rMSP1a ( Anaplasma marginale) using carbon nanotubes as carrier molecules

<div><p>Abstract Vaccination against Anaplasma marginale has been considered an important control strategy for bovine anaplasmosis. Recently, mice immunized with rMSP1 a linked to carbon nanotubes (MWNT) showed significant immune responses, generating a new possibility for use of an inactivated vaccine. The objective of this study was to investigate the cellular and humoral responses in calves immunized with MWNT+rMSP1a , associated with inactivated vaccine of A. marginale produced in vitro, and evaluate the toxic effects of the MWNT on renal and hepatic function. rMSP1a was covalently linked to MWNT. Inactivated vaccine (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. Twenty-four Holstein calves were divided (four groups) and immunized subcutaneously with PBS and non-carboxylated MWNT (control, G1), AmUFMG2 (G2), MWNT+rMSP1a (G3), and AmUFMG2 with MWNT+rMSP1a (G4). Blood samples were collected for total leukocyte counts, biochemical profiling and evaluation of the cellular and humoral response. Immunization with MWNT+rMSP1a induced increase in the total number of leukocytes, NK cells, in the lymphocyte populations and higher levels of antibodies compared to calves immunized only with AmUFMG2. Furthermore, MWNT did not induce changes in the biochemical profile. These data indicate that MWNT+rMSP1a were able to induce the immune responses more efficiently than AmUFMG2 alone, without generating toxicity.</p></div

Biomarker networks triggered by distinct immunization protocols.

<p>Network correlation analysis were assembled for pro-inflammatory and regulatory cytokines measured in supernatants from PBMCs cultures maintained upon vaccine-soluble antigen (VSA) or soluble <i>Leishmania chagasi</i> antigen (SLcA) stimuli <i>in vitro</i>. Data were analyzed at baseline before vaccination (T₀), 15 days after third immunization dose (T_3rd) as well as early (90 days—T₉₀) and late (885 days—T₈₈₅) after experimental <i>L</i>. <i>chagasi</i>-challenge. The groups are represented as follows: C (“Control”; white nodes); “Sal” (<i>Lutzomyia longipalpis</i> salivary glands; light gray nodes); “LbSal” (<i>L</i>. <i>braziliensis</i> antigen plus <i>Lutzomyia longipalpis</i> salivary glands; dark gray nodes) and “LbSapSal” (<i>L</i>. <i>braziliensis</i> antigen plus saponin and <i>Lutzomyia longipalpis</i> salivary glands; black nodes). Each connecting line represents a significant correlation between a pair of biomarkers. Dashed linesrepresent negative correlations. Solid lines represent positive correlations, and the degree of significance is represented by the line thickness [moderate correlation (continuous thin lines) for 0.370.67 or strong correlation (continuous thick lines) for r>0.68]. Spearman r indexes are used to classify the connecting edges as negative, moderate, or strong positive correlations, as shown.</p

Impact of distinct immunization protocols on the NO production elicited early and late after <i>L</i>. <i>chagasi</i>-challenge.

<p>NO levels (μM) were determined in supernatants from PBMCs cultures maintained upon vaccine-soluble antigen (VSA) or soluble <i>Leishmania chagasi</i> antigen (SLcA) stimuli <i>in vitro</i>. Data were analyzed early (90 days—T₉₀) and late (885 days—T₈₈₅) after experimental <i>L</i>. <i>chagasi</i>-challenge. The groups are represented as follows: C (“Control”; white bars); “Sal” (<i>Lutzomyia longipalpis</i> salivary glands; <i>light gray bars</i>); “LbSal” (antigen of <i>L</i>. <i>braziliensis</i> plus <i>Lutzomyia longipalpis</i> salivary glands; <i>dark gray bars</i>); and “LbSapSal” (<i>L</i>. <i>braziliensis</i> antigen plus saponin and <i>Lutzomyia longipalpis</i> salivary glands; black bars). Top panels: The x-axis displays the different experimental groups (“Control”, “Sal”, “LbSal” and “LbSapSal”) according to the <i>in vitro</i> stimuli (control culture [CC], VSA or SLcA). The y-axis represents the nitrite levels [μM]. Data are presented as mean values ± standard deviations. The connecting lines represent significant difference (<i>P <0</i>.<i>05</i>) between the CC, VSA or SLcA-stimulated cultures. The symbols C, Sal and LbSal indicate significant differences in comparison to the “Control”, “Sal” or “LbSal” groups, respectively. Bottom panels: Correlation between NO levels and spleen parasite load (# amastigotes/20ng of total DNA) at T₈₈₅ considering CC (bottom left panel) or the presence of a stimulus (VSA: bottom middle panel; or SLcA: bottom right panel) in all groups. The groups are distinguishable by colors as follows: as follows: “C” (white circles); “Sal” (ligh gray circles); “LbSal” (dark gray circles) and “LbSapSal” (black circles). The quadrants represented in the bottom panels delimit the low and high NO producers (y-axis) and the low and high spleen parasite load (x axis).</p

Impact of distinct immunization protocols on pro-inflammatory cytokine production.

<p>The levels of pro-inflammatory cytokine levels were measured in supernatants from PBMCs cultures maintained upon vaccine-soluble antigen (VSA) or soluble <i>Leishmania chagasi</i> antigen (SLcA) stimuli <i>in vitro</i>. Data were analyzed at baseline before vaccination (T₀), 15 days after third immunization dose (T_3rd) as well as early (90 days—T₉₀) and late (885 days—T₈₈₅) after experimental <i>L</i>. <i>chagasi</i>-challenge. The groups are represented as follows: C (“Control”; white bars); “Sal” (<i>Lutzomyia longipalpis</i> salivary glands; <i>light gray bars</i>); “LbSal” (antigen of <i>L</i>. <i>braziliensis</i> plus <i>Lutzomyia longipalpis</i> salivary glands; <i>dark gray bars</i>); and “LbSapSal” (<i>L</i>. <i>braziliensis</i> antigen plus saponin and <i>Lutzomyia longipalpis</i> salivary glands; black bars). The x-axis displays the different experimental groups (“Control”, “Sal”, “LbSal”, and “LbSapSal”) according to the <i>in vitro</i> stimuli (control culture [CC], VSA or SLcA). The y-axis represents the cytokine levels (pg/mL) for TNF-α (A), IL-12 (B) and IFN-γ (C). Data are presented as mean values ± standard deviations. The connecting lines represent significant difference (<i>P <0</i>.<i>05</i>) between the CC, VSA or SLcA-stimulated cultures. The symbols C, Sal and LbSal indicate significant differences in comparison to the “Control”, “Sal” and “LbSal” groups, respectively.</p

Impact of distinct immunization protocols on TGF-β production.

<p>The levels of TGF-β were measured in supernatants from PBMCs cultures maintained upon vaccine-soluble antigen (VSA) or soluble <i>Leishmania chagasi</i> antigen (SLcA) stimuli <i>in vitro</i>. Data were analyzed at baseline before vaccination (T₀), 15 days after third immunization dose (T_3rd) as well as early (90 days—T₉₀) and late (885 days—T₈₈₅) after experimental <i>L</i>. <i>chagasi</i>-challenge. The groups are represented as follows: C (“Control”; white bars) and “LbSapSal” (<i>L</i>. <i>braziliensis</i> antigen plus saponin and <i>Lutzomyia longipalpis</i> salivary glands; black bars).The x-axis displays the different experimental groups (“Control” and “LbSapSal”) according to the <i>in vitro</i> stimuli (control culture [CC] and SLcA). The y-axis represents the TGF-β levels (pg/mL). Data are presented as mean values ± standard deviations. The connecting lines represent significant difference (<i>P <0</i>.<i>05</i>) between the CC or SLcA-stimulated cultures. The symbol C indicates significant differences in comparison to the “Control” group.</p

Parasitological analysis in spleen samples late after (T₈₈₅) <i>L</i>. <i>chagasi</i>-challenge.

<p>Parasitological analysis in spleen samples late after (T₈₈₅) <i>L</i>. <i>chagasi</i>-challenge.</p

Impact of distinct immunization protocols on regulatory/anti-inflammatory cytokine production.

<p>The levels of regulatory/anti-inflammatory cytokines were measured in supernatants from PBMCs cultures maintained upon vaccine-soluble antigen (VSA) or soluble <i>Leishmania chagasi</i> antigen (SLcA) stimuli <i>in vitro</i>. Data were analyzed at baseline before vaccination (T₀), 15 days after third immunization dose (T_3rd) as well as early (90 days—T₉₀) and late (885 days—T₈₈₅) after experimental <i>L</i>. <i>chagasi</i>-challenge.The groups are represented as follows: C (“Control”; white bars); “Sal” (<i>Lutzomyia longipalpis</i> salivary glands; <i>light gray bars</i>); “LbSal” (antigen of <i>L</i>. <i>braziliensis</i> plus <i>Lutzomyia longipalpis</i> salivary glands; <i>dark gray bars</i>); and “LbSapSal” (<i>L</i>. <i>braziliensis</i> antigen plus saponin and <i>Lutzomyia longipalpis</i> salivary glands; black bars). The x-axis displays the different experimental groups (“Control”, “Sal”, “LbSal”, and “LbSapSal”) according to the <i>in vitro</i> stimuli (control culture [CC], VSA or SLcA). The y-axis represents the cytokine levels (pg/mL) for IL-4 (A) and IL-10 (B). Data are presented as mean values ± standard deviations. The connecting lines represent significant difference (<i>P <0</i>.<i>05</i>) between the CC, VSA or SLcA-stimulated cultures. The symbol Sal indicates significant differences in comparison to the “Sal” group.</p

Experimental design for simultaneous detection of anti-Trypanosomatidae IgG1 antibodies by flow cytometry-based triplex array (FC-TRIPLEX Chagas/Leish IgG1).

<p>(A) Major assay steps. PBS, phosphate-buffered saline; FBS, fetal bovine serum; PE, phycoerythrin. (B) Parasite gating strategy based on FCS versus SSC dot plot distribution followed by a set marker setup for internal control positivity limit, leading to a Percentage of Positive Fluorescent Parasites value of <2.0%. (C) This set marker setup was maintained to subsequently determine disease-related reactivity patterns. VL, visceral leishmaniasis; CH, Chagas disease; LCL, localized cutaneous leishmaniasis; NI, noninfected controls. Dark frames highlight species-specific IgG1 reactivity patterns at selected serum dilutions (VL, anti-<i>L</i>. <i>infantum</i> at 1:32,000; CH, anti-<i>T</i>. <i>cruzi</i> at 1:2,000; LCL and NI, anti-<i>L</i>. <i>braziliensis</i> at 1:1,000) according to Garcia et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref013" target="_blank">13</a>], Matos et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref015" target="_blank">15</a>], and Pereira et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref012" target="_blank">12</a>], respectively.</p

Flow cytometry-based inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity for differential diagnosis of Chagas disease and leishmaniasis.

<p>(A) Reactivity board showing the pathway for achieving distinctive disease-related positive results (triangles) and negative results (circles) results and avoiding inconclusive reactivity (squares). VL, visceral leishmaniasis; CH, Chagas disease; LCL, localized cutaneous leishmaniasis; NI, noninfected controls. (B) Proposed inverted detuned algorithm starting at the selection of the <i>L</i>. <i>infantum</i> population on FL2 versus FL4 bidimensional distribution followed by analyses of IgG1 reactivity at 1:32,000 on unidimensional FL2 histograms expressed as percentage of fluorescence positive parasites (PPFP). A PPFP value of >60% defines the VL diagnosis, whereas a PPFP value of ≤60% leads to the next algorithm step. Selection of the <i>T</i>. <i>cruzi</i> population is followed by analyses of IgG1 reactivity at 1:2,000, with a PPFP value of >50% defining the CH diagnosis, and a PPFP value of ≤50% leading to the next algorithm stage. At the final step <i>L</i>. <i>braziliensis</i> population selection is followed by analyses of IgG1 reactivity at 1:1,000, with a PPFP value of >60% defining the LCL diagnosis, and a PPFP value of ≤60% excluding all three Trypanosomatidae infections. (C) Global accuracy analyses underscored the outstanding overall performance of the method, which reached 95% (correct results were obtained for 76 of the 80 serum samples).</p

Flow cytometry-based triplex array (FC-TRIPLEX) for selective analyses of <i>L</i>. <i>infantum</i> promastigotes, <i>T</i>. <i>cruzi</i> epimastigotes, and <i>L</i>. <i>braziliensis</i> promastigotes.

<p>(A) Representative dot plots demonstrating similarities in the morphometric features of the fixed Trypanosomatidae organisms. (B) Differential fluorescence staining with fluorescein isothiocyanate (FITC) (<i>L</i>. <i>infantum</i> = 0.0 μg/mL, <i>T</i>. <i>cruzi</i> = 2.0 μg/mL, or <i>L</i>. <i>braziliensis</i> = 20.0 μg/mL) or Alexa Fluor 647 (<i>L</i>. <i>infantum</i> = 0.0 μg/mL, <i>T</i>. <i>cruzi</i> = 0.78 μg/mL, or <i>L</i>. <i>braziliensis</i> = 12.5 μg/mL) as a strategy for segregating parasites based on the FL1 and FL4 fluorometric profiles, respectively. (C) Unidimensional histograms for the FL1 and FL4 profiles illustrating the stability of fluorescently stained single or premixed parasite suspensions during storage at room temperature (RT), 4°C, or −20°C.</p