High antibody titer against apical membrane antigen-1 is required to protect against malaria in the Aotus model

Abstract

A Plasmodium falciparum 3D7 strain Apical Membrane Antigen-1 (AMA1) vaccine, formulated with AS02(A) adjuvant, slowed parasite growth in a recent Phase 1/2a trial, however sterile protection was not observed. We tested this AS02(A), 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+AS02(A) 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.

Figure 1. Recombinant 3D7 AMA1 vaccine was pure and folded correctly.

Left panel shows a coomassie blue stained SDS-PAGE analysis of the 3D7 AMA1 vaccine under non-reducing (NR) and reducing conditions (Red). Right panel shows positive reactivity of only the non-reduced 3D7 AMA1 protein with a conformational monoclonal antibody 4G2dc1.

Figure 2. The FCH/4 strain AMA1 is more homologous to 3D7 AMA1 as compared to FVO strain AMA1.

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.

Figure 3. AMA1+ISA vaccine formulation induced high titer antibodies that inhibited homologous parasite invasion.

Left panel shows ELISA endpoint titer of day-of-challenge sera and the group-mean (line). In the FCH/4 challenge group ELISA was done using 3D7 AMA1 coat antigen and for the FVO strain challenge group, ELISA was done using 3D7 and FVO AMA1 coat antigens (shown in parentheses on the x-axis). Both ELISA panels share the same y axis scale. Right panel shows the GIA activity of the day-of-challenge sera of the FCH/4 challenged groups measured at 10% serum concentration against 3D7 strain parasites.

Figure 4. Parasitemia profiles of FCH/4 and FVO challenged groups.

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.

Figure 5. 3D7 AMA1 vaccination reduced the parasite burden of the FCH/4 strain.

Mean, peak and cumulative parasitemia (parasites/µL) of individual animals and their mean (line) between days 4–11 post challenge.

Figure 6. 3D7 AMA1 vaccination slowed growth rate of FCH/4 but not FVO strain.

Group-wise mean cumulative parasitemia of the FCH/4 (left panel) and FVO (right) challenged animals plotted against days post challenge. Solid line, PBS control group; broken line, AMA+ISA; dotted line, AMA+AS02_A.

Figure 7. ELISA titers positively correlate GIA activity and negatively correlate parasitemia.

Left panel shows GIA activity of the FCH/4 challenged animals plotted against log ELISA titer. Group symbols: PBS+ISA (triangle), AMA+ISA (cross) and AMA+AS02_A (square). The three monkeys that were protected in the study had GIA activity of >70% and ELISA titer of >300,000, cut-off indicated by grey lines. Right panel shows the correlation between log ELISA endpoint titer and the cumulative day 11 parasitemia for the 18 FCH/4 challenged animals.

Figure 8. Domain-specificities of AS02_A and ISA720 induced anti-AMA1 were similar.

End-point titer was determined against chimeric proteins displaying P. falciparum domains 1, 2, 3, 1+2 or 2+3 on P. berghei 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.

Figure 9. Domain1+2 end-point titer of protected animals exceeded 100,000.

FCH/4 challenged AMA+AS02_A and AMA+ISA group titers against full-length 3D7 AMA1 (3D7) or chimeras D1, D2, D3, D1+2, D2+3 that display the corresponding P. falciparum 3D7 strain AMA1 domains on a P. berghei AMA1 scaffold (Pb). End-point titers against the P. berghei AMA1 scaffold protein are also plotted. Animals protected in the AMA+ISA group (AI-3176, AI-3179 and AI-3181) had the highest D1+2 end-point titer (red dotted line).