Plasmodium circumsporozoite protein (CSP) is a major surface antigen present in the sporozoite (Spz) stage of a malaria parasite. RTS, S vaccine, the most clinically advanced malaria vaccine, consists of a large portion of Plasmodium falciparum CSP (PfCSP). A highly infectious, recombinant rodent malaria, Plasmodium yoelii parasite bearing a full-length PfCSP, PfCSP/Py Spz, was needed as a tool to evaluate the role of PfCSP in mediating, protective, anti-malaria immunity in a mouse model.
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A transgenic parasite, PfCSP/Py Spz, was generated by inserting a construct expressing the PfCSP at the locus of the P. yoelii CSP gene by double cross-over homologous recombination. Then the biological and protective properties of PfCSP/Py Spz were determined.
This PfCSP/Py parasite produced up to 30,000 Spz in mosquito salivary glands, which is equal or even higher than the number of Spz produced by wild-type P. yoelii parasites. Five bites of PfCSP/Py-infected mosquitoes could induce blood infection in BALB/c mice.
The current study has demonstrated a successful establishment of a transgenic P. yoelii parasite clone that is able to express a full-length PfCSP, PfCSP/Py parasite. Importantly, this PfCSP/Py parasite can be as infectious as the wild-type P. yoelii parasite both in mosquito vector and in mouse, a mammalian host. A new transgenic parasite that expresses a full-length PfCSP may become a useful tool for researchers to investigate immunity against PfCSP in a mouse model.
Generation of transgenic PfCSP/Py parasites. a Transgenic parasites were generated by inserting the PfCSP expression construct at the locus of the P. yoelii CSP gene by double cross-over homologous recombination. b Correct targeting was checked by gDNA PCR. Primers used in these experiments were listed in Table 1
Approximately 300 female Anopheles stephensi mosquitoes were allowed to feed on a group of five Swiss-Webster mice infected with 0.1 % gametocyte of either wild-type P. yoelii 17XNL parasites or PfCSP/Py parasites, as previously described [25, 26]. Then, the midguts and salivary glands were dissected from a group of five infected mosquitoes from day 8 to day 26 post-infectious blood meal.
Infectivity of PfCSP/Py SPZ was determined in female BALB/c mice by two different methods. In the first method, mice were injected intravenously (iv) with 50, 150 and 450 Spz of PfCSP/Py or wild-type P. yoelii dissected from salivary glands. In the second method, mice were exposed to the bite(s) of one, five or ten PfCSP/Py parasite-infected mosquitoes. In both methods, the parasitaemia of the challenged mice was determined by microscopic examination of Giemsa-stained thin blood smears, obtained from days 3 to 10 post-Spz challenge.
Hybrid PfCSP/Py parasites produced sporozoites in mosquito. Three-hundred female Anopheles mosquitoes fed on five Swiss-Webster mice infected with wild-type P. yoelii or transgenic PfCSP/Py parasites with 0.1 % gametocytaemia. Mosquito midguts and salivary glands from five mosquitoes were dissected from day 8 to day 26 post mosquito-infectious blood meal
The complete P. falciparum life cycle cannot be maintained in vitro in the laboratory, thus limiting the experimental study of the Spz of P. falciparum human malaria parasite. Laboratory mouse malaria models have provided alternative platforms for human malaria research. Rodent malaria (P. yoelii, P. berghei, Plasmodium chabaudi, and Plasmodium vinckei) isolated from wild thicket rats have been adapted to grow in laboratory rodents. These species display many of the biological characteristics of the human malaria parasite and rodent models of malaria have been widely and successfully used to complement research on P. falciparum [30, 31].
Although recombinant rodent P. berghei parasites expressing PfCSP repeats or a full-length PfCSP were generated, their infectivity in mosquito salivary glands was lower than that of wild-type parasites. Here, P. yoelii 17XNL was used [32], which produces high number of Spz in mosquito salivary glands and is infectious to mice of both BALB/c and C57BL/6 strains [33], as the parental line for generating the PfCSP/Py transgenic parasites. The CSP, the major antigen on the Spz surface, is composed of the N-terminal flanking region, the repetitive immunodominant B-cell epitope containing central region, T-cell epitope containing C-terminal flanking region. The N- terminal regions play essential roles in parasite infectivity and could be targeted by protective antibodies [34]. In this study, a transgenic P. yoelii parasite clone that is able to express a full-length PfCSP was successfully generated and its biological properties compared to those of wild-type P. yoelii parasite were investigated.
The P. falciparum CSP has been regarded as one of the best vaccine candidates for malaria. Transgenic rodent malaria Spz expressing P. falciparum CSP represent a unique tool to evaluate PfCSP-based immunity and systematically assess formulation, molecular composition and regimen of a human malaria vaccine using a high-throughput animal experimental model. Although a recombinant rodent P. berghei parasite expressing PfCSP repeats was generated a decade ago, its infectivity in mosquito salivary glands has been modest, i.e., approximately 3000 per mosquito [8]. The infectivity of a more recently established, recombinant P. berghei parasite expressing a full-length PfCSP, is even less, i.e., only 1800 Spz are produced in the salivary gland [21]. The sequence changes introduced outside the conserved regions I and II by replacing PbCSP with PfCSP could possibly have reduced the ability of the PfCSP protein to function optimally within the P. berghei background during Spz invasion of An. stephensi salivary glands [21]. Thus, the yield of a low number of salivary gland Spz has probably hampered further PfCSP-based immunity and vaccines research, such as vaccination using irradiated PfCSP/Py Spz, which would require at least a million Spz per study.
Kumar et al. reported that the CSP is an immunodominant protective antigen in irradiated Spz [9], but Grüner et al. reported that sterile protection against malaria is independent of immune responses to the CSP [36]. In this regard, the authors are currently planning to conduct a comprehensive and independent study using heterologous immunization and challenge combinations with PfCSP/Py parasites and wild-type P. yoelii parasites in order to address this controversial issue.
In summary, the current study has demonstrated a successful establishment of a transgenic P. yoelii parasite clone that is able to express the full-length PfCSP, PfCSP/Py parasite. Importantly, this PfCSP/Py parasite can be as infectious as, or even more than, wild-type P. yoelii parasite both in mosquito vector and in mouse, a mammalian host. This indicates that the expression of the PfCSP transgene does not interfere with the infectivity and fitness of the parasite. The new transgenic parasite that expresses a full-length PfCSP may become a useful tool for researchers to investigate immunity against PfCSP in a mouse model. 2ff7e9595c
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