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

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 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

Flowchart of selection and follow-up of the Study Population from the Primary Target Sample.

<p>A total of 3,060 children, 9–12 months-old were elected for an epidemiological studies reported elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049828#pone.0049828-Collaborative1" target="_blank">[14]</a> and referred as “Primary Target Sample”. The Clinical Trial design is highlighted by dashed format. The study population enrolled in the present investigation was selected from the “Primary Target Sample” according to the PRNT results and comprise 30 PV-PRNT⁺ and 10 PV-PRNT⁻ individuals on each experimental arm (17DD and 17D-213/77), reaching a total of 80 volunteers. The current Immunological Study design is highlighted by solid format.</p

Comparative analysis of the cytokine signatures of innate and adaptive immunity triggered by the YF-17DD and YF-17D-213/77 substrains upon the <i>in vitro</i> recall of whole blood leukocytes from seroconverter children (PV-PRNT⁺) with specific YF-Ag.

<p>The diagrams highlight each leukocyte subsets with distinct tags as they display low (white rectangle) or high (black rectangle = inflammatory, gray rectangle = regulatory) cytokine producers (top panel). The ascendant frequency of volunteers with high cytokine indexes of the innate and adaptive immunity was assembled for each experimental arm and is demonstrated by bar graphs (medium panel). Comparative analysis of the overall cytokine patterns of YF-17DD (lines with black or gray triangles) and YF-17D-213/77 (lines with black or gray squares) vaccinees were further compared by overlapping the ascendant cytokine curves (bottom panel). Dotted lines highlight the 25^th and 50^th percentiles used as reference for comparative analysis. *Differences were considered relevant when the frequency for a given cytokine emerged outside the 50^th percentile as compared to the reference cytokine pattern or signature.</p

Impact of serum titers of anti-YF neutralizing antibodies on the cytokine-mediated immune response triggered by the YF-17DD and YF-17D-213/77 substrains upon <i>in vitro</i> recall of whole blood leukocytes from seroconverter children (PV-PRNT⁺) with specific YF-Ag.

<p>The PRNT⁺ groups from each experimental arm were first categorized into two subgroups referred to as PV-PRNT^MEDIUM+ (2.5≤ serum titers ≤3.5 log₁₀ mIU/mL) and PV-PRNT^HIGH+ (serum titers >3.5 log₁₀ mIU/mL). The cytokine profile of the PV-PRNT^MEDIUM+ and PV-PRNT^HIGH+ subgroups were evaluated, considering relevant the percentages of a given inflammatory cytokine that emerged higher than the 50^th percentile, as indicate by an upward arrow (↑).</p

Comparative inflammatory and regulatory cytokine signatures triggered by the YF-17DD and YF-17D-213/77 substrains upon the <i>in vitro</i> recall of whole blood leukocytes from (A) seroconverter (PV-PRNT⁺) and nonseroconverter primary vaccinees (PV-PRNT⁻) as well as (B) seroconverter revaccinees (RV-PRNT⁺) with specific YF-Ag.

<p>The ascendant frequency of volunteers with high inflammatory and regulatory cytokine indexes was assembled for each experimental arm and demonstrated by bar graphs. Comparative analysis between PV-PRNT⁺, PV-PRNT⁻, and RV-PRNT⁺ within the same experimental arm was performed taking the ascendant cytokine curve of the YF-17DD (lines with black or gray triangles) or YF-17D-213/77 (lines with black or gray squares) groups as reference. #Differences were considered relevant when the percentage of a given cytokine emerged below the quartile of the reference cytokine signatures.</p