Abstract:

Objective To investigate the truncated Plasmodium berghei putative secreted ookinete protein 7 (PbPSOP7) induces transmission-blocking immunity determined by in vitro antibody blocking and mosquito feeding assays. Methods Recombinant PbPSOP7 protein (rPbPSOP7) was expressed in E. coli and the anti-rPbPSOP7 sera were obtained by immunizing BALB/c mice for three times with rPbPSOP7 formulated with Freund’s adjuvant. For in vitro test, P. berghei-infected mouse blood was obtained from a mouse infected with 5 × 10⁶ P. berghei for three days, then co-incubated with mouse anti-rPbPSOP7 sera at 1 : 5, 1 : 10 and 1 : 50 dilutions. Mouse anti- a non-relevant His-tag protein serum at the same dilutions was used as control. The inhibition on the exflagellation of Plasmodium gametocytes was observed 15 min after the culture and the inhibition on the ookinete development was observed 24 hours after incubation. For mosquito feeding experiment, 5 mice were each immunized with 50 μg of rPbPSOP7 formulated with Freund’s adjuvant for three times, then infected with 5 × 10⁶ P. berghei by intraperitoneal injection 10 days after the last immunization. Three days post-infection, the mice were fed by at least 50 female anopheles mosquitoes. The mosquitoes were dissected 10 days after the blood feeding, the infection rate and the reduced number of oocysts in each infected mosquito were calculated compared with mosquitoes fed with mice immunized with His-tag control protein. The feeding experiment was repeated three times. Results When incubated with mouse anti-rPbPSOP7 sera at the 1 : 5, 1 : 10 or 1 : 50 dilutions for 15 min, the exflagellation of gametocytes was decreased by 22.0%, 8.0% and 2.0% compared to Plasmodium incubated with sera from mice immunized with His-tag protein(P > 0.05). After incubated with mouse anti-rPbPSOP7 sera for 24 hours, the significant inhibition of ookinete development was observed in the culture with different dilutions of the antisera. The development of ookinete was reduced by 69.2%, 56.4% and 48.6% when incubated with anti-rPbPSOP7 sera at 1 : 5, 1 : 10 and 1 : 50 dilutions, respectively, compared with control sera group (P < 0.01). Three independent mosquito feeding experiments showed that, compared with the control group, the development of oocysts in the midgut of mosquitoes in immunized group was significantly decreased from 87.1 ± 69.3, 96.4 ± 82.9 and 85.3 ± 69.1 to 31.1 ± 34.9, 30.2 ± 32.3 and 29.1 ± 35.1, respectively. Anti-rPbPSOP7 sera reduced the development of oocyst by 64.2%, 68.6% and 65.8% in three independent feeding experiments (P < 0.01). The infection rates of P. berghei in mosquitoes decreased from 96.4% (27/28), 89.3% (25/28) and 89.3% (25/28) to 78.6% (22/28), 78.6% (22/28) and 75.0% (21/28) in three experiments, with 17.8%, 10.7% and 14.3% reduction (P > 0.05). Conclusion Immunization of rPbPSOP7 induced transmission-blocking immunity characterized by reduced development of P. berghei ookinate and oocyst in mosquitoes, indicating that PbPSOP7 is a potential malaria transmission-blocking vaccine candidate.

Key words: Plasmodium berghei, Transmission-blocking vaccines, Candidate antigen, Putative secreted ookinete protein 7