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

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

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

<p>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.</p

ELISpot and CD8+ T cell IFN-γ responses of DNA/HuAd5 and HuAd5 immunized subjects to <i>P</i>. <i>falciparum</i> strains 3D7 and 7G8 AMA1 A*03 protective epitopes.

<p>ELISpot and CD8+ T cell IFN-γ activities are shown in Panels A–D. <b>Panel A:</b> ELISpot IFN-γ response of the A*03 protected subject (v11) are positive with Ap8 and the 3D7 A*03 epitope but not the 7G8 epitope (arrow). <b>Panel B:</b> ELISpot activity of v11 is not affected by CD4+-depletion but is abolished after CD8+ depletion (arrow). <b>Panel C:</b> CD8+ T cell IFN-γ responses of v11 are much higher (p = 0.001) to the 3D7 epitope than to the 7G8 epitope (arrow). <b>Panel D:</b> ELISpot IFN-γ responses of two of four non-protected subjects from the HuAd5 trial were weakly positive with the 3D7 epitope but all four subjects were negative with the 7G8 epitope (arrows).</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

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

<p>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 <i>P. falciparum</i> 3D7 strain AMA1 domains on a <i>P. berghei</i> AMA1 scaffold (Pb). End-point titers against the <i>P. berghei</i> 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).</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

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

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

Recombinant 3D7 AMA1 vaccine was pure and folded correctly.

<p>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.</p

Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

<div><p>Background</p><p>Fifteen volunteers were immunized with three doses of plasmid DNA encoding <i>P. falciparum</i> circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection.</p><p>Methodology/Principal Findings</p><p>We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to <i>P. falciparum</i> in humans.</p><p>Conclusions/Significance</p><p>We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.</p></div