Induction of strain-transcendent antibodies to placental-type isolates with VAR2CSA DBL3 or DBL5 recombinant proteins

Abstract

Background: Pregnancy associated malaria is a severe clinical syndrome associated with sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, which adheres to chondroitin sulphate A (CSA). VAR2CSA is a large and polymorphic protein that has six Duffy binding-like (DBL) domains. There is still limited understanding as to how effective individual VAR2CSA domains are at generating inhibitory antibodies or the number of domain variants needed for universal vaccine coverage.

Methods: To investigate the immunogenic properties of single domain VAR2CSA recombinant proteins, rats or rabbits were immunized with five of the six VAR2CSA domains produced in Pichia pastoris. Immune plasma was analysed against a geographically diverse panel of CSA-binding lab lines to assess antibody breadth and inhibitory activity.

Results: Of the five domains, DBL3, and to a lesser extent DBL5, induced antibodies that cross-reacted on five diverse CSA-binding parasite lines by flow cytometry. By comparison, anti-DBL6 antibodies were highly strain-specific and anti-DBL1 and anti-DBL4 antibodies were poorly reactive by flow cytometry. From this series of recombinant proteins, adhesion-blocking activity was restricted to a single rat immunized against a DBL4 recombinant protein.

Conclusions: Single domain VAR2CSA recombinant proteins produced in P. pastoris had limited efficacy in eliciting adhesion blocking antibody responses, but VAR2CSA DBL3 and DBL5 domains contain strain-transcendent epitopes that can be targeted by vaccination and may have application for vaccine development.

Figure 1

Expression of VAR2CSA-DBL recombinant proteins in P. pastoris. A) Protein schematic of VAR2CSA. The original DBL domain boundaries are indicated by black rectangles. Revised domain boundaries as described [35] are indicated in grey and numbered for the 7G8-VAR2CSA allele. The first and last amino acids of constructs are indicated below the schematic. The thin line refers to the non-var2csa encoded protein IT4var22 DBL3. B) 1 μg of His-tagged recombinant proteins were analysed under non-reducing conditions in a 4-20% SDS-PAGE gel and stained by Gel Code Blue Reagent or detected by immunoblot via anti-His tag antibodies. New 7G8-VAR2CSA and IT4var22 DBL3 recombinant proteins generated for this study are shown. Other recombinant proteins were described previously [35].

Figure 2

Endpoint titers of anti-VAR2CSA plasma as determined by ELISA. The endpoint titers of anti-VAR2CSA plasma at OD 0.1 is shown. Individual animals are indicated by dots. Mean values are indicated by bars. Rat and rabbit immune plasma were tested against their corresponding P. pastoris DBL recombinant protein at 200 ng (e.g. immune plasma/recombinant protein).

Figure 3

Cross-reactivity of anti-VAR2CSA on a panel of CSA-binding parasites. A) Flow cytometry analysis of rat anti-VAR2CSA plasma on the homologous 7G8-CSA parasite line. Preimmune plasma is shown in grey, black line for rat #1, grey for rat #2, and dashed line for rat #3. The reactivity of protein control, anti-IT4var22 DBL3 plasma is shown as well. B) Summary of homologous surface reactivity against 7G8-CSA and IT4-CSA parasite lines. Each dot represents the mean adjusted MFI from duplicate experiments observed for each animal.

Figure 4

Summary of the cross reactivity observed per DBL domain across parasite panel. The top row in each box shows the DBL domain used for immunization. On the left is listed the VAR2CSA allelic variant and animal species immunized. Mean fluorescence intensities in flow cytometry are shown as a heat map from yellow (lower) to red (higher). Rat plasma were analysed at 1/20 dilution and rabbit plasma at 1/25 dilution. Previously published observations are indicated with superscripts: ¹[30] and ²[29]. All plasma were tested against the individual HB3A and HB3B CSA-binding parasite lines except the previously published rabbit anti-IT4 DBL5 and anti-IT4 DBL6 were examined against a mixed HB3-CSA parasite line.

Figure 5

Binding inhibition activity of anti-VAR2CSA plasma. Binding inhibitory activity of anti-VAR2CSA plasma were tested in an in vitro binding assay with four different CSA-binding parasite lines and a CD36 binding control parasite line. The binding inhibitory activity was tested in a CSA-binding assay for the CSA-binding parasites and in a CD36 binding assay for the CD36 binding parasite. All anti-VAR2CSA plasma were tested as a pool of the three rats in each group, except rat #1 anti-DBL4 plasma was tested on its own because the other two rat plasma did not react by flow cytometry. A mixture of plasma against five 7G8-DBL domains (DBL1, DBL3, DBL4, DBL5, and DBL6) was also tested against the homologous 7G8-CSA parasite line. The extent of binding inhibition was expressed relative to the pool of rat immune anti-IT4var22 DBL3. The mean and range of binding inhibition from duplicate experiments is shown.