Evaluation of two sexual-stage antigens as bivalent transmission-blocking vaccines in rodent malaria

Abstract

Background: Transmission-blocking vaccine (TBV) is a promising strategy for malaria elimination. It is hypothesized that mixing or fusing two antigens targeting different stages of sexual development may provide higher transmission-blocking activity than these antigens used individually.

Methods: A chimeric protein composed of fragments of Pbg37 and PSOP25 was designed and expressed the recombinant protein in Escherichia coli Rosetta-gami B (DE3). After immunizing mice with individual recombinant proteins Pbg37 and PSOP25, mixed proteins (Pbg37+PSOP25), or the fusion protein (Pbg37-PSOP25), the antibody titers of individual sera were analyzed by ELISA. IFA and Western blot were performed to test the reactivity of the antisera with the native proteins in the parasite. The transmission-blocking activity of the different immunization schemes was assessed using in vitro and in vivo assays.

Results: When Pbg37 and PSOP25 were co-administered in a mixture or as a fusion protein, they elicited similar antibody responses in mice as single antigens without causing immunological interference with each other. Antibodies against the mixed or fused antigens recognized the target proteins in the gametocyte, gamete, zygote, and ookinete stages. The mixed proteins or the fusion protein induced antibodies with significantly stronger transmission-reducing activities in vitro and in vivo than individual antigens.

Conclusions: There was no immunological interference between Pbg37 and PSOP25. The bivalent vaccines, which expand the portion of the sexual development during which the transmission-blocking antibodies act, produced significantly stronger transmission-reducing activities than single antigens. Altogether, these data provide the theoretical basis for the development of combination TBVs targeting different sexual stages.

Keywords: Dual-antigen; Immunological interference; Transmission-blocking activity; Transmission-blocking vaccine.

Fig. 1

Expression and purification of the recombinant protein. a Diagram illustrating the expressed regions of the Pbg37, PSOP25, and their fusion Pbg37-PSOP25. The signal peptide (red box), low complexity (green box), and transmembrane region (yellow box) are highlighted. The pink line denotes the linker. b Purified recombinant Pbg37-PSOP25 (indicated by an arrow) was separated on a 10% SDS-PAGE gel and stained with Coomassie blue (left) and probed with anti-His mAb on a Western blot (right). M, PageRuler pre-stained protein ladder in kDa

Fig. 2

Analysis of specific antibodies by ELISA and Western blot. a, b BALB/c mice (n = 10) were immunized three times with Trx-His tag (immunization control) and recombinant proteins (Pbg37, PSOP25, Pbg37+PSOP25, and Pbg37-PSOP25). The equal volume sera of every mouse per group were pooled at day 10 after the final immunization for ELISA coated with the recombinant Pbg37 (a) and recombinant PSOP25 (b) polypeptides after removal of the Trx tag. Error bars indicate standard deviation. **P < 0.01 indicates a significant difference between the immunization and control groups (ANOVA). c, d The lysates of P. berghei gametocytes (GC) and ookinete (Ook) at 10 μg per lane were separated by 10% SDS-PAGE and probed with anti- Pbg37+PSOP25 antisera (c) and anti- Pbg37-PSOP25 antisera (d). The protein loading was estimated by the anti-rHsp70 sera

Fig. 3

Indirect immunofluorescence analysis using the bivalent immune sera. Parasites of different developmental stages were fixed and permeabilized with 0.1% Triton X-100. They were stained with antisera against Pbg37+PSOP25 (a) and Pbg37-PSOP25 (b) (1:200) as the primary antibodies (green). The parasites were also co-labeled with antibodies against the markers for different stages (red), including α-tubulin (α) for male gametocytes/gametes, P47 for female gametocytes, Pbs21 for zygotes and ookinetes, and SET for the nucleus of gametocytes. Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies and Alexa Fluor 555-conjugated goat anti-rabbit IgG antibodies were used as the secondary antibodies. c Negative controls showing the ookinetes labeled with the Trx-His antisera or with the secondary antibodies only. The nucleus was stained with Hoechst-33258 (blue). Images were obtained under the same conditions at a magnification of ×1000. DIC differential interference contrast microscopy. Scale bar = 5 μm

Fig. 4

Transmission-blocking activities of the antisera assessed in vitro and in vivo. a Inhibition of exflagellation. P. berghei-infected blood collected at 3 days post infection was incubated with the respective control and immune sera at dilutions of 1:5 and 1:10. Exflagellation centers in 10 microscopic fields were counted after 15 min. b) Inhibition of ookinete formation. Under the same culture conditions, ookinetes formed within 24 h were stained with Pbs21 mAb and counted. Data for exflagellation and ookinetes were representative of three independent experiments. c Midgut oocysts were counted from individual mosquitoes infected with parasites 12 days after the blood meal. The results were collected from three mice in each immunization group and in two separate experiments (n = 180). Data points represent oocyst numbers in individual mosquitoes. Horizontal bars indicate the mean number of oocysts per midgut. d Mosquito infection prevalence was calculated at 12 days after the blood meal. Data points represent the prevalence of infection in mosquitoes from three mice per group and in two separate experiments. Error bars indicate mean ± SD. *P < 0.05 and **P < 0.01 represent the significant difference between the respective immunization group and the Trx-His control group. ^#P < 0.05 and ^##P < 0.01 represent significant difference between two immunization groups