Effects of transmission-blocking vaccines simultaneously targeting pre- and post-fertilization antigens in the rodent malaria parasite Plasmodium yoelii

Abstract

Background: Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites.

Methods: We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25.

Results: Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group.

Conclusions: This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

Keywords: DNA vaccine; Malaria; Plasmodium yoelii; Pys25; Pys48/45; Transmission-blocking vaccine.

Fig. 1

The levels of specific IgGs in serum samples from BALB/c mice immunized with Pys48/45 and Pys25. The coating antigens used were lysate of purified gametocytes (a) and of cultured ookinetes (b). Results are expressed as geometric means of five in each group. Statistical analysis was performed by one-way ANOVA using the GraphPad Prism software. Asterisks (*) and (**) indicate significance at P < 0.05 and P < 0.01, respectively, as compared with the first immunization. Octothorps (#) and (##) indicate significance at P < 0.05 and P < 0.01, respectively, compared between second and third immunization. The data are representative of two separate experiments. Each mouse at the three immunization time points is coded with the same color and symbol

Fig. 2

Western blot analysis of Pys48/45 and Pys25 expression in P. yoelii gametocytes and ookinetes. Lysates from gametocyte- (a) and ookinete-enriched (b) preparations were probed with immune sera from the Pys48/45, Pys25 and Pys48/45 + Pys25 DNA immunization groups, respectively

Fig. 3

Indirect immunofluorescence assays showing the reactivity of the immune sera with P. yoelii sexual stage parasites. IFAs were performed on gametocytes (a) and ookinetes (b) with immune sera from the Pys48/45, Pys25 and Pys48/45 + Pys25 DNA immunization groups, respectively. Nuclei were counter-stained with DAPI. Scale-bars: 5 μm

Fig. 4

Transmission-blocking effect of immunization with Pys25, Pys48/45 and Pys48/45 + Pys25 plasmids in direct mosquito feeding assay. Data points represent the number of oocysts in individual mosquitoes, horizontal bars indicate the mean number of oocysts per midgut and error bars indicate SEM within individual treatments. Three independent experiments were performed. Asterisk (**) indicates significance at P < 0.01 as compared with the Vector control