VMP001-NP immunization triggers germinal center formation.

<p>(A) C57Bl/6 mice were vaccinated with 0.1 µg VMP001 and 5 µg MPLA in either soluble or VMP001-NP formulations, and on day 21, inguinal dLNs were isolated and analyzed for germinal center formation. The number of isotype-switched germinal center B cells (GL-7⁺PNA⁺), gated on B220⁺IgD^low populations in dLNs was measured with flow cytometric analysis. (B) Inguinal dLNs were cryo-sectioned on day 21 post-immunization and stained with anti-B220, anti-IgD, and anti-GL-7 (markers for B cells, immature B cells, and germinal center, respectively) and examined by confocal microscopy. Scale bars, 50 µm. *, <i>p</i><0.05, analyzed by Student's <i>t</i> test.</p

Immunization with VMP001-NPs elicits Th1/Th2 balanced antibody responses.

<p>(A) C57Bl/6 mice were immunized <i>s.c.</i> on days 0 and 21 with 25 µg of MPLA and 1 µg of VMP001 in either soluble or VMP001-NP formulations, and anti-VMP001 IgG sera were characterized on days 35 and 120 for (A) IgG, (B) IgG₁, (C) IgG_2b, (D) IgG_2c, and (E) IgG₃ titers.</p

Synthesis of PLGA NPs with surface-conjugated VMP001 (VMP001-NPs).

<p>(A) Schematic illustration of synthesis of lipid-enveloped PLGA NPs with surface-conjugated VMP001. PLGA NPs were incubated with thiolated VMP001, conjugating the antigen to maleimide-functionalized lipids displayed on the particle membranes. Particles were then PEGylated in a reaction with PEG-thiol. (B) A scanning electron microcopy image of VMP001-NPs. Scale bar, 2 µm. (C) Confocal microscopy image of VMP001-NPs incubated with anti-his-tag and fluorescent secondary antibodies to detect particle surface-conjugated VMP001. Scale bar, 10 µm.</p

VMP001-NP immunization elicits high avidity antibodies capable of agglutinating live sporozoites.

<p>(A) Avidity indices of anti-VMP001 IgG sera obtained from mice immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031472#pone-0031472-g003" target="_blank">Fig. 3</a> with soluble VMP001+MPLA (red circles) or VMP001-NP+MPLA (blue circles) were characterized over 6 months following vaccination. *, <i>p</i><0.01 and **, <i>p</i><0.001, analyzed by two-way ANOVA, followed by a Bonferroni post-test. (B) Anti-VMP001 IgG antibodies elicited with soluble VMP001+MPLA (red circles) or VMP001-NP+MPLA (blue circles) were further examined for their affinities against key fragments of VMP001, including peptides representing the Type I repeat, AGDR motif, Region I, Region II, C-terminus, and scrambled negative peptide control. Sera from non-immunized mice were also included as controls (black squares). (C,D) Sera obtain from mice on day 63 post-immunizations with 2.5 µg VMP001 and 25 µg MPLA in either (C) soluble or (D) VMP001-NP formulations were incubated with live VK210 sporozoites, and immunoflurescence assay was performed to assess recognition of native CSP present on the surface of live sporozoites by anti-VMP001 IgG sera. Mice immunized with VMP001-NP vaccines raised sera that agglutinated live VK210 subtype of <i>P. vivax</i>.</p

Social-ecological factors and preventive actions decrease the risk of dengue infection at the household-level: Results from a prospective dengue surveillance study in Machala, Ecuador

<div><p>Background</p><p>In Ecuador, dengue virus (DENV) infections transmitted by the <i>Aedes aegypti</i> mosquito are among the greatest public health concerns in urban coastal communities. Community- and household-level vector control is the principal means of controlling disease outbreaks. This study aimed to assess the impact of knowledge, attitudes, and practices (KAPs) and social-ecological factors on the presence or absence of DENV infections in the household.</p><p>Methods</p><p>In 2014 and 2015, individuals with DENV infections from sentinel clinics in Machala, Ecuador, were invited to participate in the study, as well as members of their household and members of four neighboring households located within 200 meters. We conducted diagnostic testing for DENV on all study participants; we surveyed heads of households (HOHs) regarding demographics, housing conditions and KAPs. We compared KAPs and social-ecological factors between households with (n = 139) versus without (n = 80) DENV infections, using bivariate analyses and multivariate logistic regression models with and without interactions.</p><p>Results</p><p>Significant risk factors in multivariate models included proximity to abandoned properties, interruptions in piped water, and shaded patios (p<0.05). Significant protective factors included the use of mosquito bed nets, fumigation inside the home, and piped water inside the home (p<0.05). In bivariate analyses (but not multivariate modeling), DENV infections were positively associated with HOHs who were male, employed, and of younger age than households without infections (p<0.05). DENV infections were not associated with knowledge, attitude, or reported barriers to prevention activities.</p><p>Discussion</p><p>Specific actions that can be considered to decrease the risk of DENV infections in the household include targeting vector control in highly shaded properties, fumigating inside the home, and use of mosquito bed nets. Community-level interventions include cleanup of abandoned properties, daily garbage collection, and reliable piped water inside houses. These findings can inform interventions to reduce the risk of other diseases transmitted by the <i>Ae</i>. <i>aegypti</i> mosquito, such as chikungunya and Zika fever.</p></div

Multivariate logistic regression model of predictors of acute or recent DENV infections in the household.

<p>Multivariate logistic regression model of predictors of acute or recent DENV infections in the household.</p

Social-ecological factors in households with versus without acute or recent DENV infections.

<p>Social-ecological factors in households with versus without acute or recent DENV infections.</p

KAPs in households with versus without acute or recent DENV infections.

<p>KAPs in households with versus without acute or recent DENV infections.</p

A map of the study site and distribution of study households.

<p>(A) Location of Ecuador in the Americas (B) location of the city of Machala, El Oro Province, Ecuador, (C) and the distribution of households surveyed in this study. Household locations were aggregated to the neighborhood level for de-identification. Some clusters (5 households) have been disaggregated across block boundaries. This figure was created in ArcGIS version 10.3.1 (ESRI, 2016) using shape files from the GADM database of Global Administrative Areas, version 2.8, freely available at <a href="http://gadm.org" target="_blank">gadm.org</a>. Streets are derived from data available at the OpenStreetMap project (<a href="http://openstreetmap.org" target="_blank">openstreetmap.org</a>) for the municipality of Machala, El Oro, Ecuador. Neighborhood polygons were manually digitized by AMSI, and the shape file data are available upon request to the authors.</p

Intracellular cytokine analysis shows the presence of antigen-specific cytokine production by CD4+ cells.

<p>Peripheral blood mononuclear cells from vaccinated subjects were stimulated with VMP001 and production of IL-2, IFN-γ and TNF-α was assessed. Pie chart depicts the percentage of CD4⁺ cytokine-producing cells for each cohort 2 weeks post 3^rd vaccination (Day of challenge). G, 2 and T indicate IFN-γIL-2 and TNF-α, respectively. Cytokine producers are represented by a “+” and non-producers by a “-”.</p