Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells

Abstract

Immunization with Plasmodium sporozoites that have been attenuated by gamma-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and humoral immune responses invoked by infected hepatocytes cells that contain long-lived, partially developed parasites. Here we analyzed sporozoites of Plasmodium berghei that are deficient in P36p (p36p(-)), a member of the P48/45 family of surface proteins. P36p plays no role in the ability of sporozoites to infect and traverse hepatocytes, but p36p(-) sporozoites abort during development within the hepatocyte. Immunization with p36p(-) sporozoites results in a protective immunity against subsequent challenge with infectious wild-type sporozoites, another example of a specifically genetically attenuated sporozoite (GAS) conferring protective immunity. Comparison of biological characteristics of p36p(-) sporozoites with radiation-attenuated sporozoites demonstrates that liver cells infected with p36p(-) sporozoites disappear rapidly as a result of apoptosis of host cells that may potentiate the immune response. Such knowledge of the biological characteristics of GAS and their evoked immune responses are essential for further investigation of the utility of an optimized GAS-based malaria vaccine.

Fig. 1.

Generation of the p36p^- parasite lines. (A) Schematic representation of the p36p-locus on chromosome 10 (containing p36 and the paralogue p36p) (20) and the replacement vector MI4. Correct integration of the construct results in the disrupted p36p gene as shown. Open box, untranslated regions; black box, pb36p and pb36 coding regions; gray box, tgdhfr/ts SC. (B) Disruption of p36p was shown by PCR (Right) and by Southern analysis of separated chromosomes (Left). PCR on DNA of WT and p36p^- clones (KO1 and KO2) results in the amplification of a 1.2-kb WT fragment and a 1.0-kb disrupted fragment. Chromosomes hybridized to a P. berghei (pb) dhfr/ts 3′ UTR region (DT-3′) specific probe detect the endogenous dhfr/ts copy on chromosome 7 and the integrated construct on chromosome 10. (C) The absence of p36p transcripts in p36p^- parasites as shown by RT-PCR on RNA from WT and p36p^- sporozoites with (+) or without (-) reverse transcriptase. (D) Schematic representation of the gfp insertion construct pl0019 and the cssu of the rRNA gene unit on chromosome 5 that serves as target locus for integration of the gfp vector by a single crossover. Black box, cssu; gray box, hdhfr SC; open box, gfp expression cassette. (E) Correct integration of the gfp construct in the genome of p36p^- parasites (KO1) shown by PCR (Right) and by Southern analysis of separated chromosomes (Left). PCR on DNA of WT and p36p^- gfp parasites (KOGFP) results in the amplification of a 3-kb WT fragment, a 3-kb fragment of the disrupted cssu, and a 1-kb fragment of the disrupted p36p locus. Separated chromosomes were hybridized to the DT-3′-probe, detecting pbdhfr/ts on chromosome 7, the gfp construct on chromosome 5, and the MI4 construct on chromosome 10. (F) GFP-fluorescent oocysts and sporozoites (Left) and blood stages (Right) of the p36p^-gfp parasites.

Fig. 2.

Development of WT, RAS, and p36p^- sporozoites in hepatocytes in vitro. Cells were stained by using anti-PbEXP-1 to detect the PVM, anti-HSP90, or HSP70 to visualize the parasite cytoplasm and DAPI to stain the nuclei (blue fluorescence). (A) Visualization of the PVM (green, anti-PbEXP-1) in trophozoites of WT and p36p^- parasites (red, anti-HSP90) at 15 and 24 h after infection, respectively. (B) Trophozoite development of WT (a-f), RAS (g-l), and p36p^- (m-r) parasites at 6, 12, 15, 18, 21, and 24 h after invasion using anti-HSP70 (green; bar, 10 μm).

Fig. 3.

Apoptosis is increased in p36p^- parasitized liver cells. (A) Apoptosis rates represent the percentage of parasite-invaded cells that undergo apoptosis 6 h after infection of HepG2 cells with WT, RAS, or p36p^- sporozoites. Non. Inf. indicates noninfected HepG2 cell cultures. Error bars represent SD. (B and C) Visualization of p36p^- sporozoite (green, anti-HSP70) infected hepatocytes displaying typical apoptotic signs as detected by DAPI staining (blue) in vitro (B) as well as in vivo (C). Active caspase-3 (red) detection was also performed in B. (Original magnification, ×1,260.)