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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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DBL2-X and DBL3-X expression in E. coli and purification. a. Electrophoresis in an SDS 12% polyacrylamide gel of aliquots from different steps in expression, refolding and purification of DBL3-X. Lane 1, non-induced BL21 (DE3) (containing pET24a-DBL3-X plasmid); lane 2, induced BL21 (DE3) containing the pET24a-DBL3-X plasmid; lane 3, inclusion bodies suspension; lane 4, inclusion bodies after washing and denaturation in Urea 8 M; lane 5, affinity column flow through; lane 6, portion of DBL3-X lost during refolding onto the affinity column; lane 7, affinity column elution pool; lane 8, molecular size exclusion column purified DBL3-X. b. Electrophoresis of refolded DBL3-X in a SDS 12% polyacrylamide gel before (-) and after (+) reduction with dithiothreitol (DTT). c. Electrophoresis of refolded DBL2-X in a SDS 12% polyacrylamide gel before (-) and after (+) reduction with dithiothreitol (DTT). Higher mobility before reduction indicates the presence of disulfide linkages.
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Figure 2: DBL2-X and DBL3-X expression in E. coli and purification. a. Electrophoresis in an SDS 12% polyacrylamide gel of aliquots from different steps in expression, refolding and purification of DBL3-X. Lane 1, non-induced BL21 (DE3) (containing pET24a-DBL3-X plasmid); lane 2, induced BL21 (DE3) containing the pET24a-DBL3-X plasmid; lane 3, inclusion bodies suspension; lane 4, inclusion bodies after washing and denaturation in Urea 8 M; lane 5, affinity column flow through; lane 6, portion of DBL3-X lost during refolding onto the affinity column; lane 7, affinity column elution pool; lane 8, molecular size exclusion column purified DBL3-X. b. Electrophoresis of refolded DBL3-X in a SDS 12% polyacrylamide gel before (-) and after (+) reduction with dithiothreitol (DTT). c. Electrophoresis of refolded DBL2-X in a SDS 12% polyacrylamide gel before (-) and after (+) reduction with dithiothreitol (DTT). Higher mobility before reduction indicates the presence of disulfide linkages.

Mentions: Recombinant DBL2-X and DBL3-X expressed in E. coli accumulate in inclusion bodies as insoluble, misfolded aggregates. Misfolded DBL domains were solubilized in 8 M urea, purified under denaturing conditions by metal-affinity chromatography and refolding tests were carried out by the method of rapid dilution. Using that strategy, a buffer containing 0.5% Triton-X-100 was identified as the best solution for refolding both insoluble recombinant DBL domains. However, due to the incapacity to completely remove Triton X-100 through classical methods, such as dialysis or filtration, the refolding process was done after immobilization of the denatured proteins on a metal-affinity column. In this procedure, DBL domains solubilized in 8 M urea were immobilized onto the metal-affinity column and the denaturation solution changed rapidly for the previously identified refolding solution containing Triton-X-100 (Figure 2a). Although due to sudden buffer change some DBL detached from the column, an important fraction of the protein was retained on the column. After extensive washing with the refolding buffer without the detergent, the proteins were eluted using an imidazole gradient (Figure 2a). After dialysis against 0.9% NaCl, gel filtration chromatography using Superdex 75 was performed to purify highly pure (over 95%) recombinant DBL monomers to homogeneity (Figure 2a). Using that strategy, the protein yield after refolding and all purification steps was 4 mg/l of cell culture for DBL3-X (Figure 2b) and 0.3 mg/l of cell culture for DBL2-X (Figure 2c). Due to the low yield recovered, DBL2-X was not further characterized. However, refolded and purified DBL3-X was characterized using a variety of biochemical and biophysical methods. N-terminal sequencing of recombinant DBL3-X yields the expected sequence, namely, MNATN. No other sequence was detected. Refolded DBL3-X migrated slower on SDS-PAGE gels after reduction with DTT indicating the presence of disulfide bonds (Figure 2b). Free thiol content was estimated by the method of Ellman to further examine the oxidation state of refolded DBL3-X. Free thiols can be clearly detected up to 30 μM thiol concentrations in this assay. No free thiols are detected in refolded DBL3-X at a protein concentration of 100 μM. Given that DBL3-X contains 12 cysteines, greater than 96% of cysteines are thus disulfide linked.


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)

DBL2-X and DBL3-X expression in E. coli and purification. a. Electrophoresis in an SDS 12% polyacrylamide gel of aliquots from different steps in expression, refolding and purification of DBL3-X. Lane 1, non-induced BL21 (DE3) (containing pET24a-DBL3-X plasmid); lane 2, induced BL21 (DE3) containing the pET24a-DBL3-X plasmid; lane 3, inclusion bodies suspension; lane 4, inclusion bodies after washing and denaturation in Urea 8 M; lane 5, affinity column flow through; lane 6, portion of DBL3-X lost during refolding onto the affinity column; lane 7, affinity column elution pool; lane 8, molecular size exclusion column purified DBL3-X. b. Electrophoresis of refolded DBL3-X in a SDS 12% polyacrylamide gel before (-) and after (+) reduction with dithiothreitol (DTT). c. Electrophoresis of refolded DBL2-X in a SDS 12% polyacrylamide gel before (-) and after (+) reduction with dithiothreitol (DTT). Higher mobility before reduction indicates the presence of disulfide linkages.
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Figure 2: DBL2-X and DBL3-X expression in E. coli and purification. a. Electrophoresis in an SDS 12% polyacrylamide gel of aliquots from different steps in expression, refolding and purification of DBL3-X. Lane 1, non-induced BL21 (DE3) (containing pET24a-DBL3-X plasmid); lane 2, induced BL21 (DE3) containing the pET24a-DBL3-X plasmid; lane 3, inclusion bodies suspension; lane 4, inclusion bodies after washing and denaturation in Urea 8 M; lane 5, affinity column flow through; lane 6, portion of DBL3-X lost during refolding onto the affinity column; lane 7, affinity column elution pool; lane 8, molecular size exclusion column purified DBL3-X. b. Electrophoresis of refolded DBL3-X in a SDS 12% polyacrylamide gel before (-) and after (+) reduction with dithiothreitol (DTT). c. Electrophoresis of refolded DBL2-X in a SDS 12% polyacrylamide gel before (-) and after (+) reduction with dithiothreitol (DTT). Higher mobility before reduction indicates the presence of disulfide linkages.
Mentions: Recombinant DBL2-X and DBL3-X expressed in E. coli accumulate in inclusion bodies as insoluble, misfolded aggregates. Misfolded DBL domains were solubilized in 8 M urea, purified under denaturing conditions by metal-affinity chromatography and refolding tests were carried out by the method of rapid dilution. Using that strategy, a buffer containing 0.5% Triton-X-100 was identified as the best solution for refolding both insoluble recombinant DBL domains. However, due to the incapacity to completely remove Triton X-100 through classical methods, such as dialysis or filtration, the refolding process was done after immobilization of the denatured proteins on a metal-affinity column. In this procedure, DBL domains solubilized in 8 M urea were immobilized onto the metal-affinity column and the denaturation solution changed rapidly for the previously identified refolding solution containing Triton-X-100 (Figure 2a). Although due to sudden buffer change some DBL detached from the column, an important fraction of the protein was retained on the column. After extensive washing with the refolding buffer without the detergent, the proteins were eluted using an imidazole gradient (Figure 2a). After dialysis against 0.9% NaCl, gel filtration chromatography using Superdex 75 was performed to purify highly pure (over 95%) recombinant DBL monomers to homogeneity (Figure 2a). Using that strategy, the protein yield after refolding and all purification steps was 4 mg/l of cell culture for DBL3-X (Figure 2b) and 0.3 mg/l of cell culture for DBL2-X (Figure 2c). Due to the low yield recovered, DBL2-X was not further characterized. However, refolded and purified DBL3-X was characterized using a variety of biochemical and biophysical methods. N-terminal sequencing of recombinant DBL3-X yields the expected sequence, namely, MNATN. No other sequence was detected. Refolded DBL3-X migrated slower on SDS-PAGE gels after reduction with DTT indicating the presence of disulfide bonds (Figure 2b). Free thiol content was estimated by the method of Ellman to further examine the oxidation state of refolded DBL3-X. Free thiols can be clearly detected up to 30 μM thiol concentrations in this assay. No free thiols are detected in refolded DBL3-X at a protein concentration of 100 μM. Given that DBL3-X contains 12 cysteines, greater than 96% of cysteines are thus disulfide linked.

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