Abstract:

Objective To investigate the expression and function of a transmission-blocking vaccine candidate Pb280 (PBANKA_041710) in Plasmodium berghei. Methods Homologous sequences of Plasmodium species were downloaded from NCBI and compared using MEGA7. The domains of Pb280 were predicted by SMART. Ten female Kunming (KM) mice aged 6-8 weeks were randomly selected, injected with 1 × 10⁶ P. berghei-infected red blood cells (iRBC) through tail vein. When the parasitemia in the mice reached 3%-5%, orbital blood was obtained and cultured in vitro for isolation of schizonts. The hemagglutinin (HA) tagged plasmid (Pb280HA) and Pb280 knockout plasmid (Pb280KO) were linearized and transfected to mature schizonts, which were then injected in the mice (1 × 10⁷ schizonts/mouse) through tail vein; transgenic parasites in the infected mice was identified by PCR, and underwent monoclonal screening. As a result, one strain of Pb280HA and two strains of Pb280KO (Pb280KO-C1 and Pb280KO-C2) were obtained. The expression of Pb280 in P. berghei was analyzed by Western blotting and indirect immunofluorescence assay (IFA). Another nine female KM mice were randomly divided into 3 groups (n = 3): Pb280KO-C1 group (C1 group), Pb280KO-C2 group (C2 group) and control group. Each mouse was injected with 5 × 10⁶ Pb280KO-C1, Pb280KO-C2 or wild-type (WT) P. berghei through tail vein. From day 3 to day 10 post-infection, mice tail vein blood was sampled for monitoring daily parasitemia by examining blood smears. On day 3 after infection, gametocytemia and female/male gametes ratio were calculated; mice tail vein blood was mixed with ookinete culture medium for 15 min to count the number of exflagellation centers, number of male-female integrating and number of macrogametes followed by further culture for 24 h to count the number of ookinetes. The comparisons of parasitemia, gametocytemia and female/male gamete ratio were performed by Chi-square test, and other data were analyzed with one-way ANOVA. Results Phylogenetic tree constructed by MEGA7 showed that Pb280 was genetically most close to the PY04819 of P. yoelii. SMART prediction showed that Pb280 contained an N-terminal signal peptide, 10 transmembrane domains and 1 growth factor receptor domain. Western blotting demonstrated that Pb280 protein expression occurred in P. berghei, with a relative molecular weight of 280 000. IFA results showed that Pb280 was secreted from cytoplasm to plasma membrane during the development from gametocyte to ookinete. Gene function analysis revealed that on day 3 after infection, the three groups had parasitemia of about 15%, which reached over 60% on day 10 post-infection. The parasitemia rate in the group C1 and the C2 did not differ significantly from that in the control group (P > 0.05). On day 3 post-infection, the gametocytemia rate, gamete sex ratio, number of exflagellation centers per 10 fields of view, number of male-female integrating and number of macrogametes in group C1 were 39.50‰, 1.65, 21.63 ± 4.03, 12.50 ± 8.02, and 930.00 ± 79.20, respetively; those in the group C2 were 34.50‰, 1.71, 18.25 ± 5.85, 13.75 ± 9.54, and 885.00 ± 130.11, respetively; and those in the control group were 41.50‰, 1.74, 21.44 ± 4.73, 15.31 ± 8.06, and 1 018.50 ± 58.69. There were no statistically significant differences among the three groups (P > 0.05). The numbers of ookinetes in the C1, C2 and control groups were 410.00 ± 67.88, 557.50 ± 2.12 and 782.00 ± 41.01, respectively. The numbers of ookinetes in the C1 and C2 groups did not differ significantly, but both were lower than that in the control group (P < 0.05). Conclusion Pb280 is genetically conserved in Plasmodium spp. and expressed in schizont, gametocyte and ookinete stages of P. berghei. Knockout Pb280 may result in the decrease of ookinete formation.

Key words: Malaria, Plasmodium berghei, Transmission-blocking vaccines, Pb280