Analysis of a Multi-component Multi-stage Malaria Vaccine Candidate--Tackling the Cocktail Challenge

Abstract

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.

Fig 1. Plant expression cassette and construct design, amino acid sequences, SDS-PAGE and immunoblot blot analysis.

(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 Cauliflower mosaic virus (CaMV) 35S gene; 5' untranslated region: 5'-UTR of the chalcone synthase gene from Petroselinum crispum; 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) 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.

Fig 2. Antigen-specific antibody titers of rabbit immune sera determined by ELISA.

Three rabbits (R1, R2 and R3) were immunized six times with PlasmoMix and serum samples were collected on days 0 (pre-immune), 35, 63 and 91. (A) The antibody titer against the immunization mixture (PlasmoMix) as well as against the four protein-based components (CCT, E3, gAMA1 and F0) were determined. (B) To further dissect the immune response, the specific antibody response against each individual domain was analyzed. Therefore, the domains were C-terminaly fused to DsRed (fluorescent reporter protein), and expressed and purified as described in the methods section. Antigen domains comprising a fusion protein are connected by a bracket. Antibody titers are shown for each rabbit (R1: open black square, R2: open red circle, R3: open blue triangle) as well as the geometric mean (black line) of three rabbits.

Fig 3. In vitro inhibition of (A) sporozoite gliding motility, (B) hepatocyte cell traversal, (C) sporozoite invasion and liver stage development with PlasmoMix-specific rabbit IgG and (D) Dose response of sporozoite invasion and liver stage development.

All three assays were performed with P. falciparum NF54 parasites and purified rabbit IgGs (R1, R2 and R3) at a total IgG concentration of 3 mg/ml from serum samples collected on days 63 and 91. For the dose response, purified rabbit IgGs from rabbit R3 (day 63 and day 91) was used at the following total IgG concentrations: 9, 3, 0.9 and 0.09 mg/ml. The CCT-specific antibody concentration was calculated based on CFCA from total IgG. Inhibitions are expressed as the mean of duplicate measurements with standard deviations.

Fig 4. In vitro growth inhibition assay (GIA) of asexual P. falciparum 3D7 parasites with purified PlasmoMix-specific rabbit IgG.

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 IC₅₀ 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 methods section) was used and the IC₅₀ values for gAMA1-specific antibodies were calculated. Each data point represents the mean of technical triplicates.

Fig 5. Reversal of growth inhibition assay.

The assays were performed with P. falciparum 3D7 parasites and with the intention to assess the inhibitory potential of antibodies directed against either gAMA1 or E3. The GIA was run with purified antibodies from serum samples taken on day 91 post-immunization with PlasmoMix. The assay was carried out with a total IgG concentration resulting in 40–70% inhibition in a standard GIA in the presence of either gAMA1 (A) or E3 (B) to reverse the inhibition. Each data point represents the mean with standard deviation of technical triplicates.

Fig 6. Correlation of transmission blocking activity and F0-specific antibody concentration.

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 S2 Table.