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

Boes A, Spiegel H, Voepel N, Edgue G, Beiss V, Kapelski S, Fendel R, Scheuermayer M, Pradel G, Bolscher JM, Behet MC, Dechering KJ, Hermsen CC, Sauerwein RW, Schillberg S, Reimann A, Fischer R - PLoS ONE (2015)

Bottom Line: 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.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%).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.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0131456.g001: 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.

Mentions: All cDNAs (Fig 1A and 1B) were obtained as N. benthamiana codon-optimized synthetic genes from Geneart (Invitrogen, Carlsbad, CA). The four constructs were cloned as described by Boes et al. [30] and Voepel et al. [31]. For antibody titer determination, the cDNAs coding for the single domains were fused to the C-terminus of the fluorescent reporter protein DsRed. The p19 silencing inhibitor gene (p19si) was modified as described [30]. All cloning steps were confirmed by DNA sequencing.


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

Boes A, Spiegel H, Voepel N, Edgue G, Beiss V, Kapelski S, Fendel R, Scheuermayer M, Pradel G, Bolscher JM, Behet MC, Dechering KJ, Hermsen CC, Sauerwein RW, Schillberg S, Reimann A, Fischer R - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492585&req=5

pone.0131456.g001: 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.
Mentions: All cDNAs (Fig 1A and 1B) were obtained as N. benthamiana codon-optimized synthetic genes from Geneart (Invitrogen, Carlsbad, CA). The four constructs were cloned as described by Boes et al. [30] and Voepel et al. [31]. For antibody titer determination, the cDNAs coding for the single domains were fused to the C-terminus of the fluorescent reporter protein DsRed. The p19 silencing inhibitor gene (p19si) was modified as described [30]. All cloning steps were confirmed by DNA sequencing.

Bottom Line: 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.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%).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.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany.

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.

No MeSH data available.


Related in: MedlinePlus