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Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes.

Guillotte M, Juillerat A, Igonet S, Hessel A, Petres S, Crublet E, Le Scanf C, Lewit-Bentley A, Bentley GA, Vigan-Womas I, Mercereau-Puijalon O - PLoS ONE (2015)

Bottom Line: High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain.Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational.These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité d'Immunologie Moléculaire des Parasites, Paris, France; Centre National de la Recherche Scientifique, Unité de recherche associée 2581, Paris, France.

ABSTRACT
Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.

No MeSH data available.


Related in: MedlinePlus

PfEMP1-VarO-derived antigens and immunisation schedule.(A) Coomassie blue staining of SDS-PAGE gels of the recombinant antigens used for immunisation and ELISA. The purity of the recombinant domains was assessed by electrophoresis of reduced proteins separated on 10% SDS PAGE gels. Antigens (1 μg per lane) were loaded as indicated. The molecular mass markers (M) are indicated. (B) ELISA assays of the recombinant proteins with human sera collected from 54 persons living in France and never exposed to malaria parasites (non-immune) (left panel) or individual sera from 12 Senegalese adults living in a malaria hyper-endemic setting (right panel). For details of ELISA see Materials and Methods. Please note that reactivity of individual human sera with bDBL1 is not included as it has been reported previously [39]. (C) Immunisation regimen and bleeding schedule used for all antigens.
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pone.0134292.g001: PfEMP1-VarO-derived antigens and immunisation schedule.(A) Coomassie blue staining of SDS-PAGE gels of the recombinant antigens used for immunisation and ELISA. The purity of the recombinant domains was assessed by electrophoresis of reduced proteins separated on 10% SDS PAGE gels. Antigens (1 μg per lane) were loaded as indicated. The molecular mass markers (M) are indicated. (B) ELISA assays of the recombinant proteins with human sera collected from 54 persons living in France and never exposed to malaria parasites (non-immune) (left panel) or individual sera from 12 Senegalese adults living in a malaria hyper-endemic setting (right panel). For details of ELISA see Materials and Methods. Please note that reactivity of individual human sera with bDBL1 is not included as it has been reported previously [39]. (C) Immunisation regimen and bleeding schedule used for all antigens.

Mentions: Immunisations of mice were done with native or reduced recombinant proteins using a similar regimen for all antigens (Fig 1). Unless stated otherwise, immunisation with native, soluble recombinant protein was done in groups of 5–7 inbred (BALB/c) or outbred (OF1) female mice (6–8 week old, Charles River, France), which were injected subcutaneously at 4-week intervals, with 10 μg recombinant antigens For immunisation with a reduced antigen, the protein was first reduced with 20 mM DTT for 2h at 37°C and alkylated with 0.06 M iodoacetamide for 30 min at RT. For immunisation with eDBL0 solubilised in urea, 5 inbred (BALB/c) and 5 outbred (OF1) female mice were immunised 3 times at 3-week intervals subcutaneously for the first injection and intra-muscularly for the second and third one. All antigens were mixed with Freund’s adjuvant (complete for the first immunisation and incomplete for subsequent immunisations). Sera were collected before the first immunisation (D0) and 8–10 days after the second, third or fourth injection and stored at –20°C until use.


Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes.

Guillotte M, Juillerat A, Igonet S, Hessel A, Petres S, Crublet E, Le Scanf C, Lewit-Bentley A, Bentley GA, Vigan-Womas I, Mercereau-Puijalon O - PLoS ONE (2015)

PfEMP1-VarO-derived antigens and immunisation schedule.(A) Coomassie blue staining of SDS-PAGE gels of the recombinant antigens used for immunisation and ELISA. The purity of the recombinant domains was assessed by electrophoresis of reduced proteins separated on 10% SDS PAGE gels. Antigens (1 μg per lane) were loaded as indicated. The molecular mass markers (M) are indicated. (B) ELISA assays of the recombinant proteins with human sera collected from 54 persons living in France and never exposed to malaria parasites (non-immune) (left panel) or individual sera from 12 Senegalese adults living in a malaria hyper-endemic setting (right panel). For details of ELISA see Materials and Methods. Please note that reactivity of individual human sera with bDBL1 is not included as it has been reported previously [39]. (C) Immunisation regimen and bleeding schedule used for all antigens.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4519321&req=5

pone.0134292.g001: PfEMP1-VarO-derived antigens and immunisation schedule.(A) Coomassie blue staining of SDS-PAGE gels of the recombinant antigens used for immunisation and ELISA. The purity of the recombinant domains was assessed by electrophoresis of reduced proteins separated on 10% SDS PAGE gels. Antigens (1 μg per lane) were loaded as indicated. The molecular mass markers (M) are indicated. (B) ELISA assays of the recombinant proteins with human sera collected from 54 persons living in France and never exposed to malaria parasites (non-immune) (left panel) or individual sera from 12 Senegalese adults living in a malaria hyper-endemic setting (right panel). For details of ELISA see Materials and Methods. Please note that reactivity of individual human sera with bDBL1 is not included as it has been reported previously [39]. (C) Immunisation regimen and bleeding schedule used for all antigens.
Mentions: Immunisations of mice were done with native or reduced recombinant proteins using a similar regimen for all antigens (Fig 1). Unless stated otherwise, immunisation with native, soluble recombinant protein was done in groups of 5–7 inbred (BALB/c) or outbred (OF1) female mice (6–8 week old, Charles River, France), which were injected subcutaneously at 4-week intervals, with 10 μg recombinant antigens For immunisation with a reduced antigen, the protein was first reduced with 20 mM DTT for 2h at 37°C and alkylated with 0.06 M iodoacetamide for 30 min at RT. For immunisation with eDBL0 solubilised in urea, 5 inbred (BALB/c) and 5 outbred (OF1) female mice were immunised 3 times at 3-week intervals subcutaneously for the first injection and intra-muscularly for the second and third one. All antigens were mixed with Freund’s adjuvant (complete for the first immunisation and incomplete for subsequent immunisations). Sera were collected before the first immunisation (D0) and 8–10 days after the second, third or fourth injection and stored at –20°C until use.

Bottom Line: High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain.Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational.These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité d'Immunologie Moléculaire des Parasites, Paris, France; Centre National de la Recherche Scientifique, Unité de recherche associée 2581, Paris, France.

ABSTRACT
Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.

No MeSH data available.


Related in: MedlinePlus