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Var2CSA DBL6-epsilon domain expressed in HEK293 induces limited cross-reactive and blocking antibodies to CSA binding parasites.

Fernandez P, Viebig NK, Dechavanne S, Lépolard C, Gysin J, Scherf A, Gamain B - Malar. J. (2008)

Bottom Line: Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine.Using the HEK293 expression system, DBL1-X, DBL4-epsilon and DBL6-epsilon were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-epsilon were produced at much lower levels.DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, Unité de Biologie des Interactions Hôte-Parasite, CNRS URA2581, Batiment Nicolle, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France.

ABSTRACT

Background: Pregnancy-associated malaria (PAM) is a serious consequence of Plasmodium falciparum-infected erythrocytes sequestration in the placenta through the adhesion to the placental receptor chondroitin sulfate A (CSA). Although women become resistant to PAM as they acquire transcending inhibitory immunity against CSA-binding parasites, hundreds of thousands of lives could be saved if a prophylactic vaccine targeting the surface proteins of placental parasites could be designed. Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine. However, designing such a prophylactic vaccine has been hindered by the difficulty in identifying regions of var2CSA that could elicit broadly neutralizing and adhesion-blocking antibodies.

Methods: Var2CSA is a very large protein with an estimated molecular weight of 350 kDa, and can be divided into six cysteine rich Duffy binding-like domains (DBL). The human embryonic kidney 293 cell line (HEK293) was used to produce secreted soluble recombinant forms of var2CSA DBL domains. The Escherichia coli expression system was also assessed for the domains not expressed or expressed in low amount in the HEK293 system. To investigate whether var2CSA binding DBL domains can induce biologically active antibodies recognizing the native var2CSA and blocking the interaction, mice were immunized with the refolded DBL3-X or the HEK293 secreted DBL6-epsilon domains.

Results: Using the HEK293 expression system, DBL1-X, DBL4-epsilon and DBL6-epsilon were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-epsilon were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli. Importantly, mice antisera raised against the recombinant DBL6-epsilon domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity.

Conclusion: This is the first report showing inhibitory binding antibodies obtained through a var2CSA recombinant DBL domain immunization protocol. These results support the current strategies using var2CSA as immunogen in the aim of blocking placental sequestration of malaria parasites. This work is a step towards the development of a var2CSA based vaccine that will prevent pregnancy-associated malaria and improve pregnancy outcomes.

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PEs surface recognition. a. Anti-var2CSA DBL mouse sera were tested for surface recognition of erythrocytes infected with parasites of the FCR3, 7G8 and HB3 strains with CSA- and CD36 binding phenotypes, respectively. Analysis was performed using flow cytometry and fluorescence microscopy. A representative example of a specific surface immunolabeling using the DBL6-1 antisera and the corresponding differential interferential contrast microscopy field are shown. Flow cytometry data shown are the normalized geometric mean fluorescence index (MFI) (± SD) obtained by subtracting the preimmune MFI value from the immune MFI value. b. Alignment of the DBL6-ε var2CSA sequences from the 3 parasite lines used to assess the antisera cross-reactivity by FACS. Conserved residues between FCR3 sequence and the other sequences are boxed with a yellow background.
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Figure 3: PEs surface recognition. a. Anti-var2CSA DBL mouse sera were tested for surface recognition of erythrocytes infected with parasites of the FCR3, 7G8 and HB3 strains with CSA- and CD36 binding phenotypes, respectively. Analysis was performed using flow cytometry and fluorescence microscopy. A representative example of a specific surface immunolabeling using the DBL6-1 antisera and the corresponding differential interferential contrast microscopy field are shown. Flow cytometry data shown are the normalized geometric mean fluorescence index (MFI) (± SD) obtained by subtracting the preimmune MFI value from the immune MFI value. b. Alignment of the DBL6-ε var2CSA sequences from the 3 parasite lines used to assess the antisera cross-reactivity by FACS. Conserved residues between FCR3 sequence and the other sequences are boxed with a yellow background.

Mentions: To investigate whether the recombinant var2CSA DBL domains can induce biologically active antibodies recognizing the native var2CSA, mice were immunized with the refolded DBL3 or the HEK293 secreted DBL6-ε. Final bleed antisera were evaluated for their capacity to recognize the surface of FCR3CSA binding PEs by liquid immunofluorescence and flow cytometry (Figure 3a). Two out of the three mice immunized with DBL6-ε (DBL6-1 and DBL6-3) had high level of antibodies recognizing the surface of FCR3CSA PEs. Very low reactivity was detected for mouse DBL6-2. However, in contrast to DBL6-ε, the DBL3-X antisera did not react or reacted very weakly with the surface of FCR3CSA PEs (Figure 3a).


Var2CSA DBL6-epsilon domain expressed in HEK293 induces limited cross-reactive and blocking antibodies to CSA binding parasites.

Fernandez P, Viebig NK, Dechavanne S, Lépolard C, Gysin J, Scherf A, Gamain B - Malar. J. (2008)

PEs surface recognition. a. Anti-var2CSA DBL mouse sera were tested for surface recognition of erythrocytes infected with parasites of the FCR3, 7G8 and HB3 strains with CSA- and CD36 binding phenotypes, respectively. Analysis was performed using flow cytometry and fluorescence microscopy. A representative example of a specific surface immunolabeling using the DBL6-1 antisera and the corresponding differential interferential contrast microscopy field are shown. Flow cytometry data shown are the normalized geometric mean fluorescence index (MFI) (± SD) obtained by subtracting the preimmune MFI value from the immune MFI value. b. Alignment of the DBL6-ε var2CSA sequences from the 3 parasite lines used to assess the antisera cross-reactivity by FACS. Conserved residues between FCR3 sequence and the other sequences are boxed with a yellow background.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543044&req=5

Figure 3: PEs surface recognition. a. Anti-var2CSA DBL mouse sera were tested for surface recognition of erythrocytes infected with parasites of the FCR3, 7G8 and HB3 strains with CSA- and CD36 binding phenotypes, respectively. Analysis was performed using flow cytometry and fluorescence microscopy. A representative example of a specific surface immunolabeling using the DBL6-1 antisera and the corresponding differential interferential contrast microscopy field are shown. Flow cytometry data shown are the normalized geometric mean fluorescence index (MFI) (± SD) obtained by subtracting the preimmune MFI value from the immune MFI value. b. Alignment of the DBL6-ε var2CSA sequences from the 3 parasite lines used to assess the antisera cross-reactivity by FACS. Conserved residues between FCR3 sequence and the other sequences are boxed with a yellow background.
Mentions: To investigate whether the recombinant var2CSA DBL domains can induce biologically active antibodies recognizing the native var2CSA, mice were immunized with the refolded DBL3 or the HEK293 secreted DBL6-ε. Final bleed antisera were evaluated for their capacity to recognize the surface of FCR3CSA binding PEs by liquid immunofluorescence and flow cytometry (Figure 3a). Two out of the three mice immunized with DBL6-ε (DBL6-1 and DBL6-3) had high level of antibodies recognizing the surface of FCR3CSA PEs. Very low reactivity was detected for mouse DBL6-2. However, in contrast to DBL6-ε, the DBL3-X antisera did not react or reacted very weakly with the surface of FCR3CSA PEs (Figure 3a).

Bottom Line: Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine.Using the HEK293 expression system, DBL1-X, DBL4-epsilon and DBL6-epsilon were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-epsilon were produced at much lower levels.DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, Unité de Biologie des Interactions Hôte-Parasite, CNRS URA2581, Batiment Nicolle, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France.

ABSTRACT

Background: Pregnancy-associated malaria (PAM) is a serious consequence of Plasmodium falciparum-infected erythrocytes sequestration in the placenta through the adhesion to the placental receptor chondroitin sulfate A (CSA). Although women become resistant to PAM as they acquire transcending inhibitory immunity against CSA-binding parasites, hundreds of thousands of lives could be saved if a prophylactic vaccine targeting the surface proteins of placental parasites could be designed. Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine. However, designing such a prophylactic vaccine has been hindered by the difficulty in identifying regions of var2CSA that could elicit broadly neutralizing and adhesion-blocking antibodies.

Methods: Var2CSA is a very large protein with an estimated molecular weight of 350 kDa, and can be divided into six cysteine rich Duffy binding-like domains (DBL). The human embryonic kidney 293 cell line (HEK293) was used to produce secreted soluble recombinant forms of var2CSA DBL domains. The Escherichia coli expression system was also assessed for the domains not expressed or expressed in low amount in the HEK293 system. To investigate whether var2CSA binding DBL domains can induce biologically active antibodies recognizing the native var2CSA and blocking the interaction, mice were immunized with the refolded DBL3-X or the HEK293 secreted DBL6-epsilon domains.

Results: Using the HEK293 expression system, DBL1-X, DBL4-epsilon and DBL6-epsilon were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-epsilon were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli. Importantly, mice antisera raised against the recombinant DBL6-epsilon domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity.

Conclusion: This is the first report showing inhibitory binding antibodies obtained through a var2CSA recombinant DBL domain immunization protocol. These results support the current strategies using var2CSA as immunogen in the aim of blocking placental sequestration of malaria parasites. This work is a step towards the development of a var2CSA based vaccine that will prevent pregnancy-associated malaria and improve pregnancy outcomes.

Show MeSH
Related in: MedlinePlus