Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification - Fig 2

<p>(a) Effective capture of primer on supplimer of sPIN particle. In order to see the capturing efficiency, sPIN particles were prepared with matched (red) or mismatched (black) supplimer to primer. After incubation with FAM-modified primer, both of them showed high intensity depending on the injected FAM-primer concentration (filled red and black circle). After rinsing, mismatched sPIN particle lost the fluorescence (empty black circle), meanwhile matched sPIN particle showed consistently remained fluorescence (empty red circle). It was caused by the stable binding between the primer and corresponding supplimer. (b) Positive qPCR graph of each sPIN particle using supplimers with different T_m. (c) Negative qPCR graph of each sPIN particle with supplimers of different T_m. (d) Fluorescent signal subtracted No Template Control (NTC) signal from Positive Template Control (PTC) showing regular S-shaped curves.</p

PCR reaction with sPIN particle and multiplex qPCR.

<p>PCR reaction with sPIN particle and multiplex qPCR.</p

Characteristics of <i>P</i>. <i>vivax</i>-exposed subjects from a malaria-endemic area in Thailand.

<p>Characteristics of <i>P</i>. <i>vivax</i>-exposed subjects from a malaria-endemic area in Thailand.</p

IgG isotype antibody response to PvRAMA.

<p>(A). The levels of PvRAMA- and (B) PvMSP1-19-specific antibodies of each IgG isotypes were determined in plasma from acutely <i>P</i>. <i>vivax</i>-infected patients and naïve individuals by ELISA. Vivax patients (<i>n</i> = 27) and naïve controls (<i>n</i> = 16) were randomly selected for IgG isotype prevalence studies. The cutoff value for each IgG isotype was calculated from the mean plus 2SD of the OD at 450 nm of malaria-naïve control as shown by dashed line (PvRAMA; IgG1 = 0.058, IgG2 = 0.119, IgG3 = 0.082, IgG4 = 0.051, PvMSP1-19; IgG1 = 0.011, IgG2 = 0.066, IgG3 = 0.050, IgG4 = 0.053). The significance of differences between the levels of IgG isotypes were assessed using the Mann–Whitney <i>U</i> test.</p

CD4⁺ and CD8⁺ T cell response specific for PvRAMA antigen assessed by intracellular cytokine staining and multiparameter flow cytometry.

<p>(A). Gating strategy for analysis by multiparameter flow cytometry to identify the T-cell response of PBMCs from <i>P</i>. <i>vivax</i>-recovered subjects to PvRAMA or media alone (negative control) or to phorbol myristate acetate (PMA)/ionomycin (positive control). The bar graphs represent the results obtained for PvRAMA-specific (B) IL-10-producing cells, (C) IFN-γ-producing cells and IL-2-producing cells. The significance of differences in the percentages of cytokine-producing cells were determined with the Wilcoxon matched-pairs signed-rank test.</p

Levels of naturally acquired antibody against PvRAMA in <i>P</i>. <i>vivax</i>-exposed individuals.

<p>(A). The level of PvRAMA and PvMSP1-19-specific total IgG in 77 serum samples from <i>P</i>. <i>vivax</i>-exposed individuals, including 27 samples from the acute phase, 21 samples collected 3 months after recovery, 15 samples collected 9 months after recovery, and 14 samples collected 12 months after recovery, plus 32 serum samples from malaria-naïve individuals. (B). Stability of the antibody response against PvRAMA in a group of recovered patients at different time points (acute phase, 3 months, 9 months and 12 months of recovery). The cutoff value for seropositivity was an OD greater than the mean plus two SD of that for the naïve controls (PvRAMA cutoff OD = 0.071, PvMSP1-19 cutoff OD = 0.101). Antibody levels in different groups were compared using the Mann–Whitney <i>U</i> test.</p

Stability of IgG1, IgG2 and IgG3 subclasses specific to PvRAMA antigen.

<p>The levels of IgG subclasses, (A) IgG1, (B) IgG2, (C) IgG3 in plasma of the individuals during acute infection and after 3, 9, 12 months of recovery, respectively. The cutoff value for seropositivity was an OD greater than the mean plus 2SD of that from naïve controls as shown by dashed line (IgG1 = 0.058, IgG2 = 0.119, IgG3 = 0.082, IgG4 = 0.051). IgG subclass levels in different groups were compared using the Mann-Whitney <i>U</i> test.</p

Naturally-Acquired Immune Response against <i>Plasmodium vivax</i> Rhoptry-Associated Membrane Antigen

<div><p>Rhoptry-associated membrane antigen (RAMA) is an abundant glycophosphatidylinositol (GPI)-anchored protein that is embedded within the lipid bilayer and is implicated in parasite invasion. Antibody responses against rhoptry proteins are produced by individuals living in a malaria-endemic area, suggesting the immunogenicity of <i>Plasmodium vivax</i> RAMA (PvRAMA) for induction of immune responses during <i>P</i>. <i>vivax</i> infection. To determine whether PvRAMA contributes to the acquisition of immunity to malaria and could be a rational candidate for a vaccine, the presence of memory T cells and the stability of the antibody response against PvRAMA were evaluated in <i>P</i>. <i>vivax</i>-exposed individuals. The immunogenicity of PvRAMA for the induction of T cell responses was evaluated by <i>in vitro</i> stimulation of peripheral blood mononuclear cells (PBMCs). High levels of interferon (IFN)-γ and interleukin (IL)-10 cytokines were detected in the culture supernatant of PBMCs, and the CD4⁺ T cells predominantly produced IL-10 cytokine. The levels of total anti-PvRAMA immunoglobulin G (IgG) antibody were significantly elevated, and these antibodies persisted over the 12 months of the study. Interestingly, IgG1, IgG2 and IgG3 were the major antibody subtypes in the response to PvRAMA. The frequency of IgG3 in specific to PvRAMA antigen maintained over 12 months. These data could explain the immunogenicity of PvRAMA antigen in induction of both cell-mediated and antibody-mediated immunity in natural <i>P</i>. <i>vivax</i> infection, in which IFN-γ helps antibody class switching toward the IgG1, IgG2 and IgG3 isotypes and IL-10 supports PvRAMA-specific antibody production.</p></div

Profile of IFN-γ and IL-10 in 96-h culture supernatants.

<p>PBMCs from individuals who had recovered from <i>P</i>. <i>vivax</i> infection 8–10 weeks previously were stimulated with PvRAMA antigen, media alone (negative control), or PHA (positive control) for 96 h. The concentrations of cytokines in supernatants from stimulated cells were measured by enzyme-linked immunosorbent assay (ELISA). The histograms represent the mean cytokine levels from PBMCs of ten <i>P</i>. <i>vivax</i>-recovered subjects. The significance of differences was assessed using the Wilcoxon matched-pairs signed-rank test.</p

Comparison of anti-PvRAMA antibody isotypes in <i>P</i>. <i>vivax</i>-exposed individuals and malaria-naïve individuals.

<p>Comparison of anti-PvRAMA antibody isotypes in <i>P</i>. <i>vivax</i>-exposed individuals and malaria-naïve individuals.</p