Functional antibodies against Plasmodium falciparum sporozoites are associated with a longer time to qPCR-detected infection among schoolchildren in Burkina Faso.

Background: Individuals living in malaria-endemic regions develop immunity against severe malaria, but it is unclear whether immunity against pre-erythrocytic stages that blocks initiation of blood-stage infection after parasite inoculation develops following continuous natural exposure. Methods: We cleared schoolchildren living in an area (health district of Saponé, Burkina Faso) with highly endemic seasonal malaria of possible sub-patent infections and examined them weekly for incident infections by nested PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and Liver stage antigen 1 (LSA-1). In vitro sporozoite gliding inhibition and hepatocyte invasion inhibition by naturally acquired antibodies were assessed using Plasmodium falciparum NF54 sporozoites. Associations between antibody responses, functional pre-erythrocytic immunity phenotypes and time to infection detected by 18S quantitative PCR were studied. Results: A total of 51 children were monitored. Anti-CSP antibody titres showed a positive association with sporozoite gliding motility inhibition (P<0.0001, Spearman's ρ=0.76). In vitro hepatocyte invasion was inhibited by naturally acquired antibodies (median inhibition, 19.4% [IQR 15.2-40.9%]), and there were positive correlations between invasion inhibition and gliding inhibition (P=0.005, Spearman's ρ=0.67) and between invasion inhibition and CSP-specific antibodies (P=0.002, Spearman's ρ=0.76). Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01), although this association became non-significant after adjustment for blood-stage immunity (P = 0.06). Conclusions: In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia. Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responses in vivo; epidemiological studies powered and designed to address this important question should become a research priority

Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.

BACKGROUND: A highly efficacious vaccine is needed for malaria control and eradication. Immunization with Plasmodium falciparum NF54 parasites under chemoprophylaxis (chemoprophylaxis and sporozoite (CPS)-immunization) induces the most efficient long-lasting protection against a homologous parasite. However, parasite genetic diversity is a major hurdle for protection against heterologous strains. METHODS: We conducted a double-blind, randomized controlled trial in 39 healthy participants of NF54-CPS immunization by bites of 45 NF54-infected (n = 24 volunteers) or uninfected mosquitoes (placebo; n = 15 volunteers) against a controlled human malaria infection with the homologous NF54 or the genetically distinct NF135.C10 and NF166.C8 clones. Cellular and humoral immune assays were performed as well as genetic characterization of the parasite clones. RESULTS: NF54-CPS immunization induced complete protection in 5/5 volunteers against NF54 challenge infection at 14 weeks post-immunization, but sterilely protected only 2/10 and 1/9 volunteers against NF135.C10 and NF166.C8 challenge infection, respectively. Post-immunization plasma showed a significantly lower capacity to block heterologous parasite development in primary human hepatocytes compared to NF54. Whole genome sequencing showed that NF135.C10 and NF166.C8 have amino acid changes in multiple antigens targeted by CPS-induced antibodies. Volunteers protected against heterologous challenge were among the stronger immune responders to in vitro parasite stimulation. CONCLUSIONS: Although highly protective against homologous parasites, NF54-CPS-induced immunity is less effective against heterologous parasite clones both in vivo and in vitro. Our data indicate that whole sporozoite-based vaccine approaches require more potent immune responses for heterologous protection. TRIAL REGISTRATION: This trial is registered in clinicaltrials.gov, under identifier NCT02098590

Figure S5

Distribution of immune phenotypes

Kombinierte Trochleaplastik und Rekonstruktion des MPFL bei patellofemoraler Instabilität

Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%), blood (up to 90%) and sexual parasite stages (100%). Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17-25 μg/ml), the blood stage (40-60 μg/ml) and the sexual stage (1.75 μg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy

Correlation of transmission blocking activity and F0-specific antibody concentration.

<p>The rabbit IgGs were purified from serum samples collected on day 91 post-immunization with PlasmoMix. To demonstrate the concentration dependency of the transmission blocking activity, the TBA was performed with purified antibodies from each rabbit (R1: black, R2: open and R3: hatched) at total IgG concentrations of 1 mg/ml (circles), 0.1 mg/ml (squares) and 0.01 mg/ml (triangles). Based on the CFCA results, the F0-specific antibody concentration was calculated, plotted and used to determine the IC₅₀ value (the antibody concentration needed to obtain 50% inhibition of transmission). For detailed results of the transmission blocking assay, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131456#pone.0131456.s005" target="_blank">S2 Table</a>.</p

The purified antibody preparations used for all <i>in vitro</i> assays.

<p>The total IgG concentrations and CFCA results are listed for each component. Nomenclature is based on the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). The PlasmoMix-specific antibody concentration is the sum of the specific antibody response against the four components. The antigen-specific antibody concentration is given in mg/ml and % of total IgG.</p

Antibody responses induced by CPS-CQ and CPS-MQ immunization.

<p>Antibodies against CSP (A and B; in AU), LSA-1 (C and D), and MSP-1 (E and F) were analyzed at baseline (B), 28 days after the first (I1) and second (I2) immunization and one day before challenge (C-1; 20 weeks after the last immunization) for all CPS-CQ (A, C and E, n = 5) and CPS-MQ (B, D and F, n = 10) immunized volunteers. Data are shown as individual titers with medians. Open squares indicate protected subjects, filled circles indicate unprotected subjects. Differences between the time points were analyzed by Friedman test with Dunn’s multiple comparison post-hoc test. Significant differences are indicated by asterices with * (p<0.05), ** (p<0.01), *** (p<0.001).</p

<i>In vitro</i> growth inhibition assay (GIA) of asexual <i>P</i>. <i>falciparum</i> 3D7 parasites with purified PlasmoMix-specific rabbit IgG.

<p>The rabbit IgGs were purified from the serum of three rabbits (R1, R2 and R3) collected on days 35, 63 and 91 post-immunization with PlasmoMix. Nomenclature of the sample first features the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). (A) Four serial 1/1 dilutions from 6–0.75 mg/ml of total IgGs were used to demonstrate the concentration dependency of the assay and to calculate the IC_<b>50</b> values (the total IgG concentrations needed for 50% inhibition). (B) The same GIA but instead of total IgG, the gAMA1-specifc antibody concentration (based on CFCA and calculated from total IgG, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131456#sec002" target="_blank">methods</a> section) was used and the IC_<b>50</b> values for gAMA1-specific antibodies were calculated. Each data point represents the mean of technical triplicates.</p

Plant expression cassette and construct design, amino acid sequences, SDS-PAGE and immunoblot blot analysis.

<p>(A) Schematic presentation of the expression cassettes of the plant binary expression vector pTRAk. SAR: scaffold attachment region; CaMV 35S promoter and terminator: promoter with duplicated enhancer and terminator of the <i>Cauliflower mosaic virus</i> (CaMV) 35S gene; 5' untranslated region: 5'-UTR of the chalcone synthase gene from <i>Petroselinum crispum</i>; signal peptide sequence: transit peptide sequence of murine antibody heavy chain; GOI: Gene of interest, CCT (1), gAMA1 (2), E3 (3) and F0 (4). The restriction sites used to insert the GOI into the plant expression vector are indicated; His₆ tag: six histidine affinity purification tag; ER-retention signal: SEKDEL ER-retention signal. <b>(</b>B) Table containing all the information for the selected antigens. For each antigen the main stage of expression, the name, the plasmoDB number and the amino acid sequence are depicted. SDS-PAGE (C) and immunoblot analysis (D) under reducing conditions of the four recombinant and purified proteins. Proteins were detected using rabbit anti-His₆ antiserum and alkaline phosphatase-labeled goat anti-rabbit antiserum. M: PageRuler pre-stained protein ladder (Fermentas), lane 1: CCT, lane 2: gAMA1, lane 3: E3 and lane 4: F0.</p

Adverse events during CPS immunization.

<p>Percentage of volunteers in each group experiencing possibly or probably related AE after the first (I), second (II) and third (III) immunization. AEs were evaluated at each visit and graded for severity as described in the methods paragraph: mild (light grey), moderate (dark grey) and severe (black). Only the highest intensity per subject is listed. No Serious Adverse Events occurred.</p