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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

Specificity of the antisera raised to PfEMP1 VarO-derived recombinant domains assessed by immunoblot on homologous and heterologous recombinant domains.Recombinant domains (50 ng each) were separated on 4–12% SDS gels and immunoblotted. Ten immunoblots were prepared in parallel and loaded as follows: eDBL1 (lane 1), eHead (lane 2), pCIDR (lane 3), bDBL2 (lane 4), eDBL3 (lane 5), eDBL4 (lane 6) and eDBL5 (lane 7). Blots were incubated with anti-sera against individual domains (diluted 1/ 500). (A): OF1 mouse against bDBL1; (B): OF1 mouse against pCIDR; (C): OF1 mouse against e-Head; (D): OF1 mouse against bDBL2; (E): OF1 mouse against eDBL3; (F): OF1 mouse against eDBL4; (G): OF1 mouse against eDBL5; (H): rabbit against bDBL1; (I): a pool of mouse pre-immune sera and (J): rabbit pre-immune serum.
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pone.0134292.g007: Specificity of the antisera raised to PfEMP1 VarO-derived recombinant domains assessed by immunoblot on homologous and heterologous recombinant domains.Recombinant domains (50 ng each) were separated on 4–12% SDS gels and immunoblotted. Ten immunoblots were prepared in parallel and loaded as follows: eDBL1 (lane 1), eHead (lane 2), pCIDR (lane 3), bDBL2 (lane 4), eDBL3 (lane 5), eDBL4 (lane 6) and eDBL5 (lane 7). Blots were incubated with anti-sera against individual domains (diluted 1/ 500). (A): OF1 mouse against bDBL1; (B): OF1 mouse against pCIDR; (C): OF1 mouse against e-Head; (D): OF1 mouse against bDBL2; (E): OF1 mouse against eDBL3; (F): OF1 mouse against eDBL4; (G): OF1 mouse against eDBL5; (H): rabbit against bDBL1; (I): a pool of mouse pre-immune sera and (J): rabbit pre-immune serum.

Mentions: When tested by immunoblot on the cognate and heterologous recombinant VarO domains, most sera essentially displayed domain-specific reactivity (Fig 7). However, there was some faint reaction of rabbit anti-bDBL1 with pCIDR and eDBL5 (panel H), of mouse anti-eDBL3 with pCIDR and eDBL2 (panel E) and mouse anti-bDBL2 with eDBL3 (this latter case probably reflecting the 22 amino acid sequence overlap of the DBL2 and DBL3 constructs—alignment of the various sequences is shown in S5 Fig). A stronger cross-reaction of outbred mouse anti-eDBL1 antisera with eDBL2 was observed (panel A). ELISA confirmed that antibodies raised in outbred mice to eDBL1 cross-reacted with bDBL2 and eDBL2. Antisera to bDBL1 cross-reacted less strongly with bDBL2 and eDBL2 (Fig 8). Such cross-reactivity was not observed with antibodies raised against these proteins in BALB/c mice (data not shown). Moreover it was not reciprocal, as antisera to either bDBL2 or eDBL2 failed to react with the DBL1 constructs (Fig 7 panel D and Fig 8). Thus, immunoblot and ELISA data suggest that eDBL1 and e/bDBL2, which have 27–28% protein sequence identity, share some epitopes that elicit in some animals cross-reacting antibodies when presented by the DBL1 constructs.


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)

Specificity of the antisera raised to PfEMP1 VarO-derived recombinant domains assessed by immunoblot on homologous and heterologous recombinant domains.Recombinant domains (50 ng each) were separated on 4–12% SDS gels and immunoblotted. Ten immunoblots were prepared in parallel and loaded as follows: eDBL1 (lane 1), eHead (lane 2), pCIDR (lane 3), bDBL2 (lane 4), eDBL3 (lane 5), eDBL4 (lane 6) and eDBL5 (lane 7). Blots were incubated with anti-sera against individual domains (diluted 1/ 500). (A): OF1 mouse against bDBL1; (B): OF1 mouse against pCIDR; (C): OF1 mouse against e-Head; (D): OF1 mouse against bDBL2; (E): OF1 mouse against eDBL3; (F): OF1 mouse against eDBL4; (G): OF1 mouse against eDBL5; (H): rabbit against bDBL1; (I): a pool of mouse pre-immune sera and (J): rabbit pre-immune serum.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134292.g007: Specificity of the antisera raised to PfEMP1 VarO-derived recombinant domains assessed by immunoblot on homologous and heterologous recombinant domains.Recombinant domains (50 ng each) were separated on 4–12% SDS gels and immunoblotted. Ten immunoblots were prepared in parallel and loaded as follows: eDBL1 (lane 1), eHead (lane 2), pCIDR (lane 3), bDBL2 (lane 4), eDBL3 (lane 5), eDBL4 (lane 6) and eDBL5 (lane 7). Blots were incubated with anti-sera against individual domains (diluted 1/ 500). (A): OF1 mouse against bDBL1; (B): OF1 mouse against pCIDR; (C): OF1 mouse against e-Head; (D): OF1 mouse against bDBL2; (E): OF1 mouse against eDBL3; (F): OF1 mouse against eDBL4; (G): OF1 mouse against eDBL5; (H): rabbit against bDBL1; (I): a pool of mouse pre-immune sera and (J): rabbit pre-immune serum.
Mentions: When tested by immunoblot on the cognate and heterologous recombinant VarO domains, most sera essentially displayed domain-specific reactivity (Fig 7). However, there was some faint reaction of rabbit anti-bDBL1 with pCIDR and eDBL5 (panel H), of mouse anti-eDBL3 with pCIDR and eDBL2 (panel E) and mouse anti-bDBL2 with eDBL3 (this latter case probably reflecting the 22 amino acid sequence overlap of the DBL2 and DBL3 constructs—alignment of the various sequences is shown in S5 Fig). A stronger cross-reaction of outbred mouse anti-eDBL1 antisera with eDBL2 was observed (panel A). ELISA confirmed that antibodies raised in outbred mice to eDBL1 cross-reacted with bDBL2 and eDBL2. Antisera to bDBL1 cross-reacted less strongly with bDBL2 and eDBL2 (Fig 8). Such cross-reactivity was not observed with antibodies raised against these proteins in BALB/c mice (data not shown). Moreover it was not reciprocal, as antisera to either bDBL2 or eDBL2 failed to react with the DBL1 constructs (Fig 7 panel D and Fig 8). Thus, immunoblot and ELISA data suggest that eDBL1 and e/bDBL2, which have 27–28% protein sequence identity, share some epitopes that elicit in some animals cross-reacting antibodies when presented by the DBL1 constructs.

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