Table 1.
Selected pre-erythrocytic P. falciparum proteins.
Fig 1.
Sporozoite infectivity of chimeric P. berghei lines expressing P. falciparum proteins.
Sporozoite infectivity is determined by measuring parasite liver load by in vivo imaging of luciferase-expressing liver stages at 44 hours post injection (hpi) of either 1x103 or 1x104 sporozoites and by measuring the day to blood stage patency, i.e. the day to reach 1% parasitemia. The Kaplan-Meier curves illustrate the prepatent period. Data shown from groups of 3–5 mice. (A) Chimeric P. berghei lines that showed WT-like infectivity in mice. a) Representative in vivo imaging (IVIS) of bioluminescence at 44 hpi shown in the livers of OF1 mice infected with 1x103 chimeric P. berghei sporozoites of HT, SIAP2, and RPL3. b) Parasite liver loads in mice infected with chimeric P. berghei sporozoites of HT, SIAP2 and RPL3 expressed as relative luminescence units (RLU). Significance of RLU values (Mann-Whitney test): HT—0.0159, SIAP2–0.0159 and RPL3–0.0159. c) The Kaplan-Meier curve showing the prepatent period of HT, SIAP2 and RPL3 chimeric sporozoites. Significance values of day to patency [Log-Rank (Mantel-Cox) test]: n.s. d) Representative in vivo imaging (IVIS) of bioluminescence at 44 hpi shown in the livers of OF1 mice infected with 1x104 chimeric P. berghei sporozoites of SPELD, SPATR and ETRAMP10.3. e) Parasite liver loads in mice infected with chimeric P. berghei sporozoites of SPELD, SPATR and ETRAMP10.3 expressed as relative luminescence units (RLU). Significance of RLU values (Mann-Whitney test): SPELD, SPATR and ETRAM10.3: not significant (n.s). e) The Kaplan-Meier curve showing the prepatent period of SPELD, SPATR and ETRAM10.3 chimeric sporozoites. Significance values of day to patency [Log-Rank (Mantel-Cox) test]: n.s. (B) Chimeric P. berghei lines that produced reduced or no sporozoite infectivity compared to WT sporozoites (MAEBL, GEST, SIAP1, SSP3, P36, P52, B9 and SPECT2). a) Representative in vivo imaging (IVIS) of bioluminescence at 44 hpi shown in the livers of OF1 mice infected with 1x103 chimeric P. berghei sporozoites of MAEBL, GEST, SPECT2, SSP3 and B9. b) Parasite liver loads in mice infected with chimeric P. berghei sporozoites of MAEBL, GEST and SPECT2 expressed as relative luminescence units (RLU). Significance of RLU values (Mann-Whitney test): MAEBL—0.0286, GEST (rep)—0.0159 and SPECT2–0.0079. SIAP1, SSP3, P36, P52, B9: no luminescence signal. c) Representative in vivo imaging (IVIS) of bioluminescence at 44 hpi shown in the livers of OF1 mice infected with 1x104 chimeric P. berghei sporozoites of GEST, SSP3, SIAP1, P36, P52 and Pb-PfSPECT1(r). d) The Kaplan-Meier curve showing the prepatent period of MAEBL, GEST, SSP3, SPECT2 and B9 in mice infected with 1x103 chimeric sporozoites (upper panel) and GEST, SSP3, SIAP1, P36, P52, Pb-PfSPECT2(r) and Pb-PfB9(r) in mice infected with 1x104 chimeric sporozoites (lower panel). Significance values of day to patency [Log-Rank (Mantel-Cox) test]: GEST repeat (3/5 mice)– 0.0027, GEST (1/3 mice)– 0.0253 and SSP3 (3/3 mice)– 0.0253. The other chimeric lines did not initiate blood stage infections.
Fig 2.
Schematic representation of the immunization and challenge protocol.
BALB/c and CD-1 mice were immunized with ChAd63- and MVA viral-vectored P. falciparum proteins using a prime boost protocol followed by challenge with 1x103 chimeric P. berghei sporozoites expressing P. falciparum proteins. The plot on the right shows visualization of the number of naïve or immunized mice that show no or protected immunity (either no blood stage infection or delay in the pre-patency) after challenge with the chimeric parasite expressing the cognate antigen. Protective immunity is determined by measuring the day to blood stage patency i.e., the day to reach 1% parasitemia.
Fig 3.
Expression of P. falciparum proteins in chimeric P. berghei sporozoites.
Immunofluorescence analysis demonstrating P. falciparum protein expression in chimeric P. berghei sporozoites. Salivary-gland sporozoites of the different chimeric lines were stained with sera from the corresponding vaccinated mice (Alexa Fluor 488, green) and Hoechst-33342 (blue; nuclear staining). As a control, wild-type (WT) P. berghei sporozoites were stained with the same anti-sera. Merged images of the different channels are shown for both chimeric and WT P. berghei sporozoite stained images.
Fig 4.
Protective immunity of viral-vectored P. falciparum antigens.
Protective immunity in mice immunized with ChAd63- and MVA viral-vectored P. falciparum proteins as determined by the (delay in) the day to blood stage patency, i.e. the day to reach 1% parasitemia after challenge with chimeric P. berghei sporozoites expressing the P. falciparum proteins. Eight immunized and eight naïve BALB/c mice and ten immunized and ten naïve CD-1 mice were challenged with 103 chimeric sporozoites by intravenous injection. The Kaplan-Meier curves illustrate the time to 1% parasitaemia and statistical significance between the survival curves was assessed using the Log-Rank (Mantel-Cox) Test (group size of inbred BALB/c mice: n = 6–8; group size of CD-1 outbred mice: n = 9–10. (a) PfHT p = 0.48 (BALB/c) and p = 0.77 (CD-1), (b) PfRPL3 p = 0.43 (BALB/c) and p = 0.86 (CD-1), (c) PfSPELD p = 0.003 (BALB/c) and p = 0.004 (CD1), (d) PfETRAMP10.3 p = 0.14 (BALB/c) and p = 0.38 (CD-1), (e) PfSIAP2 p = 0.48 (BALB/c) and p = 0.20 (CD-1), and (f) PfSPATR p = 0.73 (BALB/c) and p = 0.25 (CD-1).