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The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

Nielsen MA, Resende M, de Jongh WA, Ditlev SB, Mordmüller B, Houard S, Ndam NT, Agerbæk MØ, Hamborg M, Massougbodji A, Issifou S, Strøbæk A, Poulsen L, Leroy O, Kremsner PG, Chippaux JP, Luty AJ, Deloron P, Theander TG, Dyring C, Salanti A - PLoS ONE (2015)

Bottom Line: Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies.In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences.Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies.

View Article: PubMed Central - PubMed

Affiliation: Centre for Medical Parasitology at Department of Immunology and Microbiology, Faculty of Health and Medical Science, University of Copenhagen, and Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark.

ABSTRACT
The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a broadly strain-transcending antibody response.

No MeSH data available.


Related in: MedlinePlus

Comparison of the induction of binding inhibitory antibodies against antigens expressed in High-Five or S2 cells.Bars indicate the mean binding inhibition of 3D7 (A) or FCR3 (B) Plasmodium falciparum infected erythrocytes (IE). The values are the percentages of released IEs in wells with test serum compared to wells with control serum from non-immunized animals. Serum was used in a one to ten dilution. Error bars indicate the coefficients of variation of the mean of triplicate measurements (100*(SD/(mean of control binding)).
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pone.0135406.g001: Comparison of the induction of binding inhibitory antibodies against antigens expressed in High-Five or S2 cells.Bars indicate the mean binding inhibition of 3D7 (A) or FCR3 (B) Plasmodium falciparum infected erythrocytes (IE). The values are the percentages of released IEs in wells with test serum compared to wells with control serum from non-immunized animals. Serum was used in a one to ten dilution. Error bars indicate the coefficients of variation of the mean of triplicate measurements (100*(SD/(mean of control binding)).

Mentions: One of the challenges of recombinant expression of PfEMP1 molecules is the structural complexity of the antigens, thus several expression systems have been explored previously [19]. In previous pre-clinical development efforts we have worked with High-Five cells using the baculovirus expression vector system [20]. Insect cells enable expression of large proteins with multiple disulfide bonds without the need for refolding. As the baculovirus system, from a regulatory point of view, is not ideal for cGMP production we transitioned antigen expression to cGMP qualified S2 cells using a plasmid transfection system proprietary to ExpreS2ion Biotechnologies, thus avoiding virus infection. As this was a previously unexplored expression system for malaria antigens, we wanted to evaluate this transition in terms of the functional antibody response. Originally the plan was to make head-to-head comparisons of antibodies raised by immunizations with all antigens expressed in S2 or High-Five cells. However, a direct comparison of antigens was only possible for eight antigen pairs (Table 1 and Fig 1), as not all antigens could be expressed in both systems (Data not shown). The in vitro efficacy of antibody-specific inhibition of homologous IE binding to CSA was used to evaluate the potency of the vaccine and showed equivalence in the immunogenicity of antigens expressed in High-Five cells or in S2 cells (Fig 1)(Wilcoxon: P = 0.9).


The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

Nielsen MA, Resende M, de Jongh WA, Ditlev SB, Mordmüller B, Houard S, Ndam NT, Agerbæk MØ, Hamborg M, Massougbodji A, Issifou S, Strøbæk A, Poulsen L, Leroy O, Kremsner PG, Chippaux JP, Luty AJ, Deloron P, Theander TG, Dyring C, Salanti A - PLoS ONE (2015)

Comparison of the induction of binding inhibitory antibodies against antigens expressed in High-Five or S2 cells.Bars indicate the mean binding inhibition of 3D7 (A) or FCR3 (B) Plasmodium falciparum infected erythrocytes (IE). The values are the percentages of released IEs in wells with test serum compared to wells with control serum from non-immunized animals. Serum was used in a one to ten dilution. Error bars indicate the coefficients of variation of the mean of triplicate measurements (100*(SD/(mean of control binding)).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556615&req=5

pone.0135406.g001: Comparison of the induction of binding inhibitory antibodies against antigens expressed in High-Five or S2 cells.Bars indicate the mean binding inhibition of 3D7 (A) or FCR3 (B) Plasmodium falciparum infected erythrocytes (IE). The values are the percentages of released IEs in wells with test serum compared to wells with control serum from non-immunized animals. Serum was used in a one to ten dilution. Error bars indicate the coefficients of variation of the mean of triplicate measurements (100*(SD/(mean of control binding)).
Mentions: One of the challenges of recombinant expression of PfEMP1 molecules is the structural complexity of the antigens, thus several expression systems have been explored previously [19]. In previous pre-clinical development efforts we have worked with High-Five cells using the baculovirus expression vector system [20]. Insect cells enable expression of large proteins with multiple disulfide bonds without the need for refolding. As the baculovirus system, from a regulatory point of view, is not ideal for cGMP production we transitioned antigen expression to cGMP qualified S2 cells using a plasmid transfection system proprietary to ExpreS2ion Biotechnologies, thus avoiding virus infection. As this was a previously unexplored expression system for malaria antigens, we wanted to evaluate this transition in terms of the functional antibody response. Originally the plan was to make head-to-head comparisons of antibodies raised by immunizations with all antigens expressed in S2 or High-Five cells. However, a direct comparison of antigens was only possible for eight antigen pairs (Table 1 and Fig 1), as not all antigens could be expressed in both systems (Data not shown). The in vitro efficacy of antibody-specific inhibition of homologous IE binding to CSA was used to evaluate the potency of the vaccine and showed equivalence in the immunogenicity of antigens expressed in High-Five cells or in S2 cells (Fig 1)(Wilcoxon: P = 0.9).

Bottom Line: Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies.In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences.Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies.

View Article: PubMed Central - PubMed

Affiliation: Centre for Medical Parasitology at Department of Immunology and Microbiology, Faculty of Health and Medical Science, University of Copenhagen, and Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark.

ABSTRACT
The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a broadly strain-transcending antibody response.

No MeSH data available.


Related in: MedlinePlus