In Vitro Antiparasitic Effect of the Volunteers' Antibodies in ADCI Assay

<p>Shown are results obtained with volunteers' serum samples collected either at month 5 (A) or at month 12 (B), as compared to the African immune IgG pool able to transfer clinical protection in humans (dark bars, pool of immune African globulin-positive control). Each bar represents the mean value obtained with each volunteer serum, in three separate experiments ± SD. The results from WB assays (performed with months 5 and 12 samples side by side with a positive control) are shown below those of the ADCI assay for each individual volunteer and are expressed as either negative (−) or positive (+ or ++). For each group, the increasing grey colour corresponds to increasing immunisation doses, e.g., from left to right, Montanide (unhatched bars) 10–10–10, 20–20–20, 30–30–10, and 100–10–10, and for alum (hatched bars) 30–30–30 and 100–10–10. SGI values 30% or greater are considered positive. Dotted line indicates the threshold of positivity of the ADCI assay.</p

Mean Biological Effect of Antibodies in Either Direct or Monocyte-Dependent Fashion, at Various Time Points with Each Adjuvant

<p>Shown are the means ± standard error of the mean of the effects of sera from all volunteers in direct growth inhibition assays (used as a control in each ADCI assay; see Methods) and in monocyte-dependent ADCI assays (sera from 30 volunteers were analyzed at each time point, i.e., the figure summarizes results from 90 sera). Triangles, Montanide-adjuvated vaccine; circles, alum-adjuvated vaccine. Open symbols, direct growth inhibition by antibodies; solid symbols, monocyte-dependent ADCI assays. Months 0, 5, and 12: sera collected before immunisation, 1 mo after the last immunisation, and 12 mo after the first immunisation, respectively.</p

In Vivo Passive Transfer of the Volunteers' Antibodies in <i>P. falciparum–</i>Infected Humanised Mice

<div><p>Shown are representative examples of results obtained by passive transfer of Western Blot positive sera collected at month 5 (A), or of control sera (B), and of WB-positive sera collected at month 12 (C).</p> <p>(A) <i>P. falciparum</i> infected SCID mice received 200 μl of sera delivered IP from three WB-positive volunteers, collected at month 5, 1 mo after the last immunisation. Shown are results from two mice that received, first, normal monocytes (MN), then monocytes with preimmunisation control sera (month 0), followed by month 5 sera with monocytes (solid arrows corresponding to volunteers 14 and 16, open and solid squares, respectively), one mouse receiving first monocytes followed by monocytes with month 5 serum (dotted arrows, dotted line, open circles)</p> <p>(B) <i>P. falciparum–</i>infected SCID mice received 200 μl of sera from controls. Either monocytes followed by monocytes with serum from a WB-negative volunteer (dotted arrows, dotted line, open circles), or monocytes with preimmunisation samples from two volunteers followed by serum alone, repeated twice (plain arrows, solid and open squares).</p> <p>(C) <i>P. falciparum–</i>infected SCID mice received 200 μl of sera from three WB-positive volunteers, collected at month 12. All animals received monocytes first, followed by monocytes with the 12-mo serum, followed by serum alone. Reproducibility is shown in two animals receiving the serum from a single donor (volunteer 21, solid squares and open circles). Transfer of serum alone was ineffective (solid squares, days 6 and 7) indicating that the strong in vivo antiparasitic effect depends on monocyte-antibody cooperation.</p></div

Immunogenicity of the MSP3-LSP in Volunteers Receiving the Vaccine Adjuvated by Montanide or Alum

<div><p>(A) Scheme of immunisation (arrows) and of sampling (plain circles). Samples for immunoassays were taken 1 mo after each immunisation.</p> <p>(B) Lymphoproliferative responses (bars) and IFN-γ secretion (*), ± SD, as compared to controls. PHA, phytohemagglutinin; TT, tetanus toxoid. IFN-γ values for TT and PHA are those obtained using month 5 samples.</p> <p>(C) Mean ELISA IgG titres to the MSP3-LSP at various time points during and after immunisation (months 1, 5, and 12 after the first immunisation).</p> <p>(D) Proportion of WB-positive individuals in each group at different time points ± 95% confidence intervals.</p> <p>(E) Isotype distribution of antibodies measured in ELISA with IgG subclass-specific secondary antibodies (data from samples collected at month 5).</p> <p>In each graph, the increasing grey colour corresponds to increasing immunisation doses, e.g., for Montanide (unhatched bars) from left to right, 10–10–10, 20–20–20, 30–30–10, 100–10–10, and for alum (hatched bars) 30–30–30 and 100–10–10.</p></div