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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

Immunofluorescence assay with live infected or formalin-fixed PaloAlto VarO erythrocytes.Live Palo Alto VarO mature infected erythrocytes (pigmented trophozoites and schizonts) (A-D) or formalin-fixed cultures (E) were incubated with mouse antisera against PfEMP1-VarO-derived recombinant domains at 1/200 dilution, and bound antibodies visualised using Alexa Fluor 488-labelled goat anti-mouse IgG (Invitrogen) at 1/1,000 dilution and nuclei were labelled with Hoechst 33342 dye at 1/1,000. Typical punctate surface staining of the membrane of the infected erythrocyte (green) was observed with antisera raised to bDBL1, eDBL1, pCIDR, eDBL2, bDBL2, eDBL4 and eHead (representative examples are shown here in (B-D) as indicated. Antisera raised to eDBL0, which failed to react with the erythrocyte surface, reacted with a perinuclear image on formalin-fixed Palo Alto VarO parasites (E). Pre-immune sera to formalin-fixed parasites were negative (not shown). The parasite nuclei are stained with DAPI (blue). Slides were viewed with a 100x objective using a Leica CTR5000 fluorescent microscope (35).
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pone.0134292.g006: Immunofluorescence assay with live infected or formalin-fixed PaloAlto VarO erythrocytes.Live Palo Alto VarO mature infected erythrocytes (pigmented trophozoites and schizonts) (A-D) or formalin-fixed cultures (E) were incubated with mouse antisera against PfEMP1-VarO-derived recombinant domains at 1/200 dilution, and bound antibodies visualised using Alexa Fluor 488-labelled goat anti-mouse IgG (Invitrogen) at 1/1,000 dilution and nuclei were labelled with Hoechst 33342 dye at 1/1,000. Typical punctate surface staining of the membrane of the infected erythrocyte (green) was observed with antisera raised to bDBL1, eDBL1, pCIDR, eDBL2, bDBL2, eDBL4 and eHead (representative examples are shown here in (B-D) as indicated. Antisera raised to eDBL0, which failed to react with the erythrocyte surface, reacted with a perinuclear image on formalin-fixed Palo Alto VarO parasites (E). Pre-immune sera to formalin-fixed parasites were negative (not shown). The parasite nuclei are stained with DAPI (blue). Slides were viewed with a 100x objective using a Leica CTR5000 fluorescent microscope (35).

Mentions: Construct eDBL0, devoid of the NTS region, was produced as inclusion bodies, solubilised in 3 M urea, 10 mM DTT and injected in a denatured form. It was quite immunogenic, as indicated by high ELISA reactivity with the native eDBL1 and bDBL1 constructs (S4A and S4B Fig, Table 2). The individual response of BALB/c mice was somewhat scattered, with 50% ELISA titres ranging from 1/25,000 to 1/125,000 on eDBL1, and even more scattered when assessed against bDBL1, with 50% ELISA titres ranging from 1/5,000 to 1/75,000. The protein was more immunogenic in OF1 outbred mice (50% ELISA titres consistently above 1/625,000 for 4/5 animals, data not shown), i.e. in the same range as outbred mice immunised with the native eDBL1 or bDBL1 (Table 2). The sera reacted with PfEMP1-VarO on immunoblots of reduced Palo Alto iRBCs, but failed to react with the non—reduced extract (Fig 4A and 4B, lane 8). The sera also failed to react by surface IFA (Fig 5B) and to prevent or interfere with VarO rosetting (Fig 5E and Table 2). Interestingly, the sera reacted by IFA on fixed parasite smears, producing a perinuclear image, consistent with a reaction with the ER-enclosed, unfolded protein (Fig 6). We conclude that this denatured N-terminal domain, truncated of its NTS region induces antibodies reacting with PfEMP1 epitopes that are not displayed on the iRBC surface.


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)

Immunofluorescence assay with live infected or formalin-fixed PaloAlto VarO erythrocytes.Live Palo Alto VarO mature infected erythrocytes (pigmented trophozoites and schizonts) (A-D) or formalin-fixed cultures (E) were incubated with mouse antisera against PfEMP1-VarO-derived recombinant domains at 1/200 dilution, and bound antibodies visualised using Alexa Fluor 488-labelled goat anti-mouse IgG (Invitrogen) at 1/1,000 dilution and nuclei were labelled with Hoechst 33342 dye at 1/1,000. Typical punctate surface staining of the membrane of the infected erythrocyte (green) was observed with antisera raised to bDBL1, eDBL1, pCIDR, eDBL2, bDBL2, eDBL4 and eHead (representative examples are shown here in (B-D) as indicated. Antisera raised to eDBL0, which failed to react with the erythrocyte surface, reacted with a perinuclear image on formalin-fixed Palo Alto VarO parasites (E). Pre-immune sera to formalin-fixed parasites were negative (not shown). The parasite nuclei are stained with DAPI (blue). Slides were viewed with a 100x objective using a Leica CTR5000 fluorescent microscope (35).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134292.g006: Immunofluorescence assay with live infected or formalin-fixed PaloAlto VarO erythrocytes.Live Palo Alto VarO mature infected erythrocytes (pigmented trophozoites and schizonts) (A-D) or formalin-fixed cultures (E) were incubated with mouse antisera against PfEMP1-VarO-derived recombinant domains at 1/200 dilution, and bound antibodies visualised using Alexa Fluor 488-labelled goat anti-mouse IgG (Invitrogen) at 1/1,000 dilution and nuclei were labelled with Hoechst 33342 dye at 1/1,000. Typical punctate surface staining of the membrane of the infected erythrocyte (green) was observed with antisera raised to bDBL1, eDBL1, pCIDR, eDBL2, bDBL2, eDBL4 and eHead (representative examples are shown here in (B-D) as indicated. Antisera raised to eDBL0, which failed to react with the erythrocyte surface, reacted with a perinuclear image on formalin-fixed Palo Alto VarO parasites (E). Pre-immune sera to formalin-fixed parasites were negative (not shown). The parasite nuclei are stained with DAPI (blue). Slides were viewed with a 100x objective using a Leica CTR5000 fluorescent microscope (35).
Mentions: Construct eDBL0, devoid of the NTS region, was produced as inclusion bodies, solubilised in 3 M urea, 10 mM DTT and injected in a denatured form. It was quite immunogenic, as indicated by high ELISA reactivity with the native eDBL1 and bDBL1 constructs (S4A and S4B Fig, Table 2). The individual response of BALB/c mice was somewhat scattered, with 50% ELISA titres ranging from 1/25,000 to 1/125,000 on eDBL1, and even more scattered when assessed against bDBL1, with 50% ELISA titres ranging from 1/5,000 to 1/75,000. The protein was more immunogenic in OF1 outbred mice (50% ELISA titres consistently above 1/625,000 for 4/5 animals, data not shown), i.e. in the same range as outbred mice immunised with the native eDBL1 or bDBL1 (Table 2). The sera reacted with PfEMP1-VarO on immunoblots of reduced Palo Alto iRBCs, but failed to react with the non—reduced extract (Fig 4A and 4B, lane 8). The sera also failed to react by surface IFA (Fig 5B) and to prevent or interfere with VarO rosetting (Fig 5E and Table 2). Interestingly, the sera reacted by IFA on fixed parasite smears, producing a perinuclear image, consistent with a reaction with the ER-enclosed, unfolded protein (Fig 6). We conclude that this denatured N-terminal domain, truncated of its NTS region induces antibodies reacting with PfEMP1 epitopes that are not displayed on the iRBC surface.

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