High Antibody Titer against Apical Membrane Antigen-1 Is Required to Protect against Malaria in the Aotus Model

A Plasmodium falciparum 3D7 strain Apical Membrane Antigen-1 (AMA1) vaccine, formulated with AS02A adjuvant, slowed parasite growth in a recent Phase 1/2a trial, however sterile protection was not observed. We tested this AS02A, and a Montanide ISA720 (ISA) formulation of 3D7 AMA1 in Aotus monkeys. The 3D7 parasite does not invade Aotus erythrocytes, hence two heterologous strains, FCH/4 and FVO, were used for challenge, FCH/4 AMA1 being more homologous to 3D7 than FVO AMA1. Following three vaccinations, the monkeys were challenged with 50,000 FCH/4 or 10,000 FVO parasites. Three of the six animals in the AMA+ISA group were protected against FCH/4 challenge. One monkey did not become parasitemic, another showed only a short period of low level parasitemia that self-cured, and a third animal showed a delay before exhibiting its parasitemic phase. This is the first protection shown in primates with a recombinant P. falciparum AMA1 without formulation in Freund's complete adjuvant. No animals in the AMA+AS02A group were protected, but this group exhibited a trend towards reduced growth rate. A second group of monkeys vaccinated with AMA+ISA vaccine was not protected against FVO challenge, suggesting strain-specificity of AMA1-based protection. Protection against FCH/4 strain correlated with the quantity of induced antibodies, as the protected animals were the only ones to have in vitro parasite growth inhibitory activity of >70% at 1∶10 serum dilution; immuno-fluorescence titers >8,000; ELISA titers against full-length AMA1 >300,000 and ELISA titer against AMA1 domains1+2 >100,000. A negative correlation between log ELISA titer and day 11 cumulative parasitemia (Spearman rank r = −0.780, p value = 0.0001), further confirmed the relationship between antibody titer and protection. High titers of cross-strain inhibitory antibodies against AMA1 are therefore critical to confer solid protection, and the Aotus model can be used to down-select future AMA1 formulations, prior to advanced human trials

Domain-specificities of AS02_A and ISA720 induced anti-AMA1 were similar.

<p>End-point titer was determined against chimeric proteins displaying <i>P. falciparum</i> domains 1, 2, 3, 1+2 or 2+3 on <i>P. berghei</i> AMA1 scaffold (D1, D2, D3, D1+2, D2+3 chimeras respectively). Domain-specific titer was calculated by expressing the domain-specific end-point titer as a percentage of titer against the full-length 3D7 AMA1 protein. Mean and standard error for 6 animals per group was plotted.</p

The FCH/4 strain AMA1 is more homologous to 3D7 AMA1 as compared to FVO strain AMA1.

<p>Top panel shows the amino acid differences between 3D7, FVO and FCH/4 AMA1. Polymorphisms within the grey cells are included in the sequence boundary of 3D7 AMA1 vaccine, amino acid 83–531. The disulphide bonded domains (D1, D2 and D3) are marked by thick lines. D1 (amino acid 95–300), D2 (308–404) and D3 (439–584). Lower panel shows the crystal structure of AMA1 (amino acids 97–531) with the location of the 3D7-FVO (left) and 3D7-FCH/4 (right) amino acid differences shown as solid balls (D1 polymorphisms - red; D2 - green and D3 polymorphisms - blue). The residues of the C1 cluster (187, 190, 196, 197, 200, 204, 206 and 225) are circled in green.</p

Immunogenicity and efficacy data of individual animals.

<p>Vaccine and challenge groups, animal ID, ELISA endpoint titer against 3D7 or FVO AMA1 coated plates, IFA titer against 3D7 and FVO schizonts, GIA activity against <i>P. falciparum</i> 3D7 or FVO target parasite and parasite burden (mean, peak and cumulative counts) between days 4 and 11 post challenge are shown. ** Two animals in the PBS+ISA_FVO group died due to unrelated causes during the vaccination phase. GIA was not done (nd). The ELISA titer values are 1000X.</p

Parasitemia profiles of FCH/4 and FVO challenged groups.

<p>Daily parasitemia of animals plotted against days-post-challenge (days). Solid lines represent a virulent parasitemia profile that required drug treatment for high parasitemia (>200,000/µL) within 15 days of challenge. Broken lines represent a slower progressing and self-limiting infection profile. The challenge strains are indicated in parentheses (FCH/4 or FVO). Two monkeys in the AMA+ISA_FCH/4 group (AI3181, AI-3176) were re-challenged on day 16.</p