A Malaria Vaccine That Elicits in Humans Antibodies Able to Kill Plasmodium falciparum

BACKGROUND: Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults) that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant. METHODS AND FINDINGS: Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum–infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation. CONCLUSION: This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory

Human Recombinant Antibodies against Plasmodium falciparum Merozoite Surface Protein 3 Cloned from Peripheral Blood Leukocytes of Individuals with Immunity to Malaria Demonstrate Antiparasitic Properties

Immunoglobulins from individuals with immunity to malaria have a strong antiparasitic effect when transferred to Plasmodium falciparum malaria infected patients. One prominent target of antiparasitic antibodies is the merozoite surface antigen 3 (MSP-3). We have investigated the antibody response against MSP-3 residues 194 to 257 (MSP-3(194-257)) on the molecular level. mRNA from peripheral blood leukocytes from clinically immune individuals was used as a source of Fab (fragment antibody) genes. A Fab-phage display library was made, and three distinct antibodies designated RAM1, RAM2, and RAM3 were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3 full-length antibodies have been produced in CHO cells. Reactivity with the native parasite protein was demonstrated by immunofluorescence microscopy, flow cytometry, and immunoblotting. Furthermore, the antiparasitic effect of RAM1 has been tested in vitro in an antibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 and the IgG3 versions of the antibody show an inhibitory effect on parasite growth

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

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

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