Development of a Plasmodium berghei transgenic parasite expressing the full-length Plasmodium vivax circumsporozoite VK247 protein for testing vaccine efficacy in a murine model

The experimental protocols used and all care and handling of the animals were in accordance with the guidelines for animal care and use prepared by Kanazawa University (Ref. no. 22118–1) and Obihiro University of Agriculture and Veterinary Medicine (Ref. no. 26–109).

HepG2 cells were maintained as described previously [19]. The monoclonal antibodies (mAbs) used were as follows: 2F2, mAb specific for the repeat sequence [DRA(D/A)GQPAG] of PvCSP(VK210) (MRA-184; Malaria Research and Reference Reagent Resource Center [MR4], Manassas, VA, USA); 2E10E9, mAb specific for the repeat sequence (ANGAGNQPG) of PvCSP(VK247) (MRA-185; MR4); 3D11, mAb specific for the repeat sequence of PbCSP of the ANKA strain (PbCSP) (MRA-100; MR4).

To construct the transfer plasmid for generating the PvCSP(VK247)/Pb transgenic parasite line in place of native PbCSP, the DNA sequence corresponding to amino acids His₂₄-Asp₃₇₀ of the entire PvCSP VK247 gene (GenBank accession number M69059) minus its signal peptide and glycosylphosphatidylinositol (GPI)-anchor sequence was amplified from pEU3-PvCSP(VK247) using pPvCSP-VK247-F4 and pPvCSP-VK247-R1 primers (Table 1). The PCR product was then cloned into pENTR (Invitrogen life Technologies, Carlsbad, CA, USA) to construct pENTR-PvCSP-VK247-MunI. Our previous study confirmed that the GPI anchor of the PfCSP moieties derived from PvCSP(VK210) were able to function in the transgenic rodent parasites [18]. The DNA sequence corresponding to amino acids Lys₃₇₂-Asn₃₉₅ of the GPI anchor of PfCSP (Accession number AAN87615) was amplified from pBS-5′UTR-PfCSP-Tcell [20] using pPvCSP-VK247-F1 and pPfCSP-R7 primers (Table 1), and then cloned into pENTR to construct the pENTR-PvCSP-VK247-XmaI/MunI plasmid. A 1.1-kb fragment of PvCSP(VK247) was excised from pENTR-PvCSP-VK247-MunI by digestion with XmaI and MunI and then inserted into the XmaI/MunI sites of pENTR-PvCSP-VK247-XmaI/MunI to construct pENTR-PvCSP-VK247-R1. A 1.7-kb fragment of the PvCSP(VK247) nucleotide sequence and GPI anchor nucleotide sequence of PfCSP was excised from pENTR-PvCSP-VK247-R1 by digestion with XmaI and SfuI and then inserted into the XmaI/SfuI sites of pBS-5′UTR-PvCSP(VK210)-dihydrofolate reductase (DHFR)-3′ [18] to construct pBS-5′UTR-PvCSP(VK247)-DHFR-3′-R1. The transgenic PvCSP(VK247)/Pb parasite was generated by transfection of WT-Pb with the linearized pBS-5′UTR-PvCSP(VK247)-DHFR-3′ plasmid, as described previously [21]. For genotype analysis, genomic PvCSP(VK247)/Pb DNA was extracted from PvCSP(VK247)/Pb-infected mouse blood using a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). The replacement of Pbcsp gene with the Pvcsp gene was confirmed by PCR using a set of forward and reverse gene-specific primers (Table 1).

Sporozoites were separated by 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to an Immobilon-FL PVDF membrane (Merck Millipore, Guyancourt, France) and then probed with either of the following mAbs: 3D11, 2F2, or 2E10E9. Blots probed with the appropriate secondary Abs conjugated to IRDye 800 (Rockland Immunochemicals, Gilbertsville, PA, USA) with the same membranes were visualized using an Odyssey infrared imager (LI-COR, Lincoln, NE, USA).

Sporozoites isolated from mosquito salivary glands were loaded onto glass slides and fixed as described previously [18]. Each slide was stained with an Alexa Fluor 488-conjugated 2E10E9 mAb and mounted with a drop of VECTASHIELD containing 4′, 6′-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). An LSM710 inverted laser scanning microscope (Carl Zeiss, Tokyo, Japan) was used for image acquisition.

Anopheles stephensi mosquitoes (SDA 500 strain) were allowed to feed either on WT-Pb, PvCSP(VK210)/Pb, or PvCSP(VK247)/Pb parasite-infected mice. The infectivity of these parasites from mice to mosquitoes was assessed at day 14 post-feeding. Mosquito midguts were dissected in sterile PBS and stained with mercury chromate solution as described previously [21], after which the prevalence and numbers of oocysts were recorded. Sporozoite development in the mosquito salivary glands was assessed on day 21 after the blood meal was taken. Mice, bitten by three to seven mosquitoes infected with either PvCSP(VK210)/Pb or PvCSP(VK247)/Pb were checked for the development of blood-stage parasites by microscopic examination of Giemsa-stained thin blood smears.

HepG2 cells were seeded at a density of 5 × 10⁴ cells/well in a collagen type I-coated 48-well plate 48 h prior to the addition of PvCSP(VK247)/Pb sporozoites. Sporozoites (2 × 10³), isolated from mosquito salivary glands and then incubated with 100-fold dilutions of 2F2 or 2E10E9 mAbs, were added to the HepG2 cells. The sporozoites were incubated with the cells for 72 h, the culture media was changed every 24 h, and total RNA was extracted from the cells using a QIAamp RNA blood mini kit (Qiagen). First-strand cDNA synthesis was carried out using MultiScribe™ reverse transcriptase (Applied Biosystems, Life Technologies, Foster City, CA, USA) with random primers. Semi-quantitative PCR was performed with P. berghei 18s rRNA and human β-actin primers (Table 1). P. berghei 18s rRNA copy numbers reflect the exoerythrocytic forms (EEF) numbers in the HepG2 cells [22].

Using a plasmid encoding the full-length Pvcsp(VK247) gene and the dihydrofolate reductase (DHFR) gene (which confers resistance to pyrimethamine) to transfect the parasites, the PvCSP(VK247)/Pb parasite was generated (Fig. 1). Briefly, the full-length Pvcsp(VK247) gene consists of the regions encoding the N-terminal signal peptide and C-terminal GPI anchor, both of which are derived from Pfcsp. The P. vivax replacement cassette represents the CSP sequence of the P. vivax VK247 strain, from amino acids 24–370. WT-Pb blood stage parasites were transfected in vitro with a plasmid linearized by the XhoI restriction enzyme, as described previously [21]. The transfected parasites, injected intravenously into BALB/c mice, were selected under pyrimethamine treatment. Ten days later, drug-resistant parasites were recovered and cloned by limiting dilution.

Gene replacement by genetic recombination and genomic integration of the Pvcsp(VK247) gene cassette was confirmed in the transgenic parasite, PvCSP(VK247)/Pb (Fig. 2a). The western blotting data showed clearly that the PvCSP(VK247)/Pb sporozoites stained positive with the 2E10E9 mAb and negative with the 3D11 mAb or the 2F2 mAb (Fig. 2b). The relative molecular mass (Mr) of the PvCSP(VK247) protein is 37.0 kDa (Fig. 2b), a value lower than its predicted molecular weight (~50 kDa), and possibly resulting from cleavage of the precursor because cleavage of CSP occurs on the sporozoite surface when parasites contact hepatocytes [23, 24]. No cross-reactivity was observed between the 2E10E9 mAb and either WT-Pb, PvCSP(VK210)/Pb or PvCSP(VK247)/Pb sporozoites. IFAs showed that the PvCSP(VK247) protein was expressed on the surface of each sporozoite isolated from the salivary glands of PvCSP(VK247)/Pb-infected mosquitoes (Fig. 2c).

Oocyst and sporozoite counts for the PvCSP(VK247)/Pb transgenic parasite were compared with those of the WT-Pb or the transgenic PvCSP(VK210)/Pb parasite lines. On day 14 post feeding, the PvCSP(VK247)/Pb-infected mosquito midguts contained approximately 150 healthy-looking oocysts per midgut. Oocyst intensity and prevalence in the PvCSP(VK247)/Pb line were not reduced compared with those of the WT-Pb or the PvCSP(VK210)/Pb parasite lines (Table 2; Fig. 3a). On day 21, after dissecting the mosquito salivary glands, the total number and percentage prevalence of sporozoites from PvCSP(VK247)/Pb were comparable to those of the WT-Pb and the PvCSP(VK247)/Pb lines (Table 2; Fig. 3b). To examine whether PvCSP(VK247)/Pb transgenic parasites had adapted to their in vivo environment, BALB/c mice were challenged through the biting of three to seven PvCSP(VK247)/Pb- or PvCSP(VK210)/Pb-infected mosquitoes. At 5 and 7 days post-infection, the parasite prevalence and parasitaemias of the subsequent blood-stage infections were monitored. Although the percentage of the infected mice differed between the PvCSP(VK210)/Pb and the PvCSP(VK247)/Pb lines on day 5, there was no statistically significant difference between them (Fig. 4). All the mice (7/7) became infected 7 days after being bitten by PvCSP(VK247)/Pb- or PvCSP(VK210)/Pb-infected mosquitoes. There was no significant difference between the parasitaemias attained by the mice from either parasite line on day 5 and 7. Thus, these results indicate that the PvCSP(VK247)/Pb and the PvCSP(VK210)/Pb lines were comparable in terms of their parasitaemias and infectivity profiles in mice. Figures 3 and 4 show that the PvCSP(VK247)/Pb parasite line behaved sufficiently similar to the WT-Pb or the PvCSP(VK210)/Pb lines in the mosquito and the mouse stages to allow it to be used for measuring the protective efficacy of CSP-based vaccines.

To evaluate the utility of the transgenic PvCSP(VK247)/Pb parasite line as a tool for assessing the efficacy of future vaccines, the neutralization capabilities of anti-PvCSP mAbs in vitro were tested. Figure 5 shows that PvCSP(VK247)/Pb sporozoite invasion of HepG2 cells was inhibited by the 2E10E9 mAb, but not by the 2F2 mAb. This result indicates that PvCSP(VK247)/Pb sporozoite invasion of HepG2 cells is inhibited by the mAb targeting the PvCSP repeat region of VK247.