Antibody Responses to a Novel Plasmodium falciparum Merozoite Surface Protein Vaccine Correlate with Protection against Experimental Malaria Infection in Aotus Monkeys

The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals

High-Level Expression of the Malaria Blood-Stage Vaccine Candidate Plasmodium falciparum Apical Membrane Antigen 1 and Induction of Antibodies That Inhibit Erythrocyte Invasion

Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression of Plasmodium falciparum FVO strain AMA-1 (PfAMA-1) in Pichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites in Plasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologous P. falciparum by immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologous P. falciparum

Statistical Model To Evaluate In Vivo Activities of Antimalarial Drugs in a Plasmodium cynomolgi-Macaque Model for Plasmodium vivax Malaria▿ ‡

Preclinical animal models informing antimalarial drug development are scarce. We have used asexual erythrocytic Plasmodium cynomolgi infections of rhesus macaques to model Plasmodium vivax during preclinical development of compounds targeting parasite phospholipid synthesis. Using this malaria model, we accumulated data confirming highly reproducible infection patterns, with self-curing parasite peaks reproducibly preceding recrudescence peaks. We applied nonlinear mixed-effect (NLME) models, estimating treatment effects in three drug studies: G25 (injected) and the bisthiazolium prodrugs TE4gt and TE3 (oral). All compounds fully cured P. cynomolgi-infected macaques, with significant effects on parasitemia height and time of peak. Although all three TE3 doses tested were fully curative, NLME models discriminated dose-dependent differential pharmacological antimalarial activity. By applying NLME modeling treatment effects are readily quantified. Such drug development studies are more informative and contribute to reduction and refinement in animal experimentation

A. Heat map of antibody reactivity to FVO Block 2 serotype peptides over the course of immunization. MSP-1 Block 2 specific peptide ELISA is as described in Figure 4 and in Materials and Methods. Reactivities of sera from immunized <i>Aotus</i> are shown as blue rectangles for each peptide tested, with darker colored bars indicating higher ELISA reactivity as shown in the figure key. Columns represent serum reactivity for each time point, and each panel shows reactivity for all pre-challenge samples from each animal. B. Amino acid sequence of the FVO MSP-1 Block 2 antigen. MSP-1 Block 2 flanking sequences are shown in red and internal repeat sequences in blue, matching the peptide sequences shown in Panel A.

<p>A. Heat map of antibody reactivity to FVO Block 2 serotype peptides over the course of immunization. MSP-1 Block 2 specific peptide ELISA is as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083704#pone-0083704-g004" target="_blank">Figure 4</a> and in Materials and Methods. Reactivities of sera from immunized <i>Aotus</i> are shown as blue rectangles for each peptide tested, with darker colored bars indicating higher ELISA reactivity as shown in the figure key. Columns represent serum reactivity for each time point, and each panel shows reactivity for all pre-challenge samples from each animal. B. Amino acid sequence of the FVO MSP-1 Block 2 antigen. MSP-1 Block 2 flanking sequences are shown in red and internal repeat sequences in blue, matching the peptide sequences shown in Panel A.</p

Schedule of immunization, blood sampling and parasite challenge of <i>Aotus lemurinus griseimembra</i>.

<p>The day of sampling and/or immunization is shown with the appropriate week of each time point shown in brackets.</p><p>i.v. injection (1 x 10⁵<i>P. falciparum</i> FVO parasites - ring stage).</p><p>Parasite challenge Go/No Go decision point, based on IFA titer.</p><p>Drug treatment criteria: Parasitemia ≥5% and/or haematocrit ≤20%.</p

<i>P. falciparum</i> parasitaemia development in all challenged <i>Aotus</i>.

<p>Percentage parasitaemia is plotted against day of sampling post challenge. Open symbols, immunized animals; filled symbols, control (naïve, non-immunized) animals.</p

Reactivity of <i>Aotus</i> sera with parasite proteins.

<p>Schizont extracts from the Wellcome (W) and 3D7 (3) isolates were probed by Western blotting with sera from all four immunized animals. Serum samples from day 97 (pre-challenge) and day 120 (post challenge) from each animal were tested in parallel on contiguous parts of the same membrane. Immunized animal code numbers are shown on the left of each panel. Arrowheads indicate reactivity with the N-terminal p83 proteolytic fragment of MSP-1. The dominant 50 kDa band in all blots is the heavy chain of human IgG, recognized by the secondary reagent (HRP conjugated anti-human IgG heavy chain).</p