Non-Apical Membrane Antigen 1 (AMA1) IgGs from Malian Children Interfere with Functional Activity of AMA1 IgGs as Judged by Growth Inhibition Assay

BACKGROUND: Apical membrane antigen 1 (AMA1) is one of the best-studied blood-stage malaria vaccine candidates. When an AMA1 vaccine was tested in a malaria naïve population, it induced functionally active antibodies judged by Growth Inhibition Assay (GIA). However, the same vaccine failed to induce higher growth-inhibitory activity in adults living in a malaria endemic area. Vaccination did induce functionally active antibodies in malaria-exposed children with less than 20% inhibition in GIA at baseline, but not in children with more than that level of baseline inhibition. METHODS: Total IgGs were purified from plasmas collected from the pediatric trial before and after immunization and pools of total IgGs were made. Another set of total IgGs was purified from U.S. adults immunized with AMA1 (US-total IgG). From these total IgGs, AMA1-specific and non-AMA1 IgGs were affinity purified and the functional activity of these IgGs was evaluated by GIA. Competition ELISA was performed with the U.S.-total IgG and non-AMA1 IgGs from malaria-exposed children. RESULTS: AMA1-specific IgGs from malaria-exposed children and U.S. vaccinees showed similar growth-inhibitory activity at the same concentrations. When mixed with U.S.-total IgG, non-AMA1 IgGs from children showed an interference effect in GIA. Interestingly, the interference effect was higher with non-AMA1 IgGs from higher titer pools. The non-AMA1 IgGs did not compete with anti-AMA1 antibody in U.S.-total IgG in the competition ELISA. CONCLUSION: Children living in a malaria endemic area have a fraction of IgGs that interferes with the biological activity of anti-AMA1 antibody as judged by GIA. While the mechanism of interference is not resolved in this study, these results suggest it is not caused by direct competition between non-AMA1 IgG and AMA1 protein. This study indicates that anti-malaria IgGs induced by natural exposure may interfere with the biological effect of antibody induced by an AMA1-based vaccine in the target population

Phase 1 Trial of Malaria Transmission Blocking Vaccine Candidates Pfs25 and Pvs25 Formulated with Montanide ISA 51

Pfs25 and Pvs25, surface proteins of mosquito stage of the malaria parasites P. falciparum and P. vivax, respectively, are leading candidates for vaccines preventing malaria transmission by mosquitoes. This single blinded, dose escalating, controlled Phase 1 study assessed the safety and immunogenicity of recombinant Pfs25 and Pvs25 formulated with Montanide ISA 51, a water-in-oil emulsion.The trial was conducted at The Johns Hopkins Center for Immunization Research, Washington DC, USA, between May 16, 2005-April 30, 2007. The trial was designed to enroll 72 healthy male and non-pregnant female volunteers into 1 group to receive adjuvant control and 6 groups to receive escalating doses of the vaccines. Due to unexpected reactogenicity, the vaccination was halted and only 36 volunteers were enrolled into 4 groups: 3 groups of 10 volunteers each were immunized with 5 microg of Pfs25/ISA 51, 5 microg of Pvs25/ISA 51, or 20 microg of Pvs25/ISA 51, respectively. A fourth group of 6 volunteers received adjuvant control (PBS/ISA 51). Frequent local reactogenicity was observed. Systemic adverse events included two cases of erythema nodosum considered to be probably related to the combination of the antigen and the adjuvant. Significant antibody responses were detected in volunteers who completed the lowest scheduled doses of Pfs25/ISA 51. Serum anti-Pfs25 levels correlated with transmission blocking activity.It is feasible to induce transmission blocking immunity in humans using the Pfs25/ISA 51 vaccine, but these vaccines are unexpectedly reactogenic for further development. This is the first report that the formulation is associated with systemic adverse events including erythema nodosum.ClinicalTrials.gov NCT00295581

Immunogenicity of Self-Associated Aggregates and Chemically Cross-Linked Conjugates of the 42 kDa Plasmodium falciparum Merozoite Surface Protein-1

Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP142) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP142 conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP142 self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP142 specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis

Impact on Malaria Parasite Multiplication Rates in Infected Volunteers of the Protein-in-Adjuvant Vaccine AMA1-C1/Alhydrogel+CPG 7909

BACKGROUND: Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. METHODS: In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. RESULTS: A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson r = -0.93 [95% CI: -1.0, -0.27] P = 0.02) and AMA1 antibody titres in the vaccine group (Pearson r = -0.93 [95% CI: -0.99, -0.25] P = 0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] P = 0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5-9], control group median 9 days [range 7-9]). CONCLUSIONS: Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. TRIAL REGISTRATION: ClinicalTrials.gov [NCT00984763]

The Epitope of Monoclonal Antibodies Blocking Erythrocyte Invasion by <em>Plasmodium falciparum</em> Map to The Dimerization and Receptor Glycan Binding Sites of EBA-175

<div><p>The malaria parasite, <em>Plasmodium falciparum</em>, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by <em>Plasmodium falciparum</em> by preventing formation of the EBA-175– glycophorin A complex.</p> </div

Effect of arginine mutation on EBA-175 RII binding to human erythrocytes.

a<p>the rosette number was an average counted from two biological repeats in each experiment.</p>b<p>erythrocyte-binding assay was performed with normal erythrocytes in two independent experiments.</p>c<p>erythrocyte-binding assay was performed with neuraminidase-treated erythrocytes Nm, neuraminidase.</p>*<p>Residue 566 of EBA-175 corresponds to residue 422 of rEBA-175 RII.</p

Deduced amino acid sequences of clones enriched for each EBA-175 RII specific mAb.

<p>Deduced amino acid sequences of clones enriched for each EBA-175 RII specific mAb.</p

F2βf peptide competes with mAbs R215 (A) and R217 (B) but not R218 (C) for binding to recombinant EBA-175 RII by Surface Plasmon Resonance studies.

<p>Monoclonal antibody sensograms show binding to immobilized EBA-175 RII with the concentrations of mAb and cyclic F2βf peptide as indicated. The concentrations of cyclic F2βf peptide were added to a fixed concentration of mAb.</p

Mapping of epitope predictions to the rEBA-175 RII crystal structure.

<p>(A) rEBA-175 RII crystal structure as a dimer of two RII molecules (cyan and grey), the region corresponding to the F2βf peptide is highlighted in yellow and R422 is highlighted in magenta. The amino- and carboxy- terminal residues are colored in blue and red respectively. The white box represents the region highlighted in panel B. (B) The F2 domain of one monomer in the rEBA-175 RII crystal structure is shown as a ribbon diagram and residues are colored by the number of times they were predicted by either PepSurf and Mapitope to be part of the epitope for mAbs R215, R217, or R256 from never (cyan) to most often (magenta; see SOM for raw data). Residues discussed in the text are labeled according to rEBA-175 RII numbering (EBA-175 3D7 sequence number –144) and shown in stick representation.</p

Summary of binding properties of EBA-175 RII specific mAbs.

<p>SPR: surface plasmon resonance. ND: not determined.</p>*<p>Does not inhibit parasite growth <i>in vitro</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056326#pone.0056326-Sim2" target="_blank">[21]</a>.</p>**<p>None of the mAbs demonstrated binding to linear F2βf.</p>c<p>These mAbs were shown to compete against each other for binding by competition ELISA.</p