| Malaria is still one of the most devastating diseases all over the world, with 50% population at risk and about 1-3 billion new cases every year, resulting in 1-2 million deaths annually. Effective vaccine would highly reduce the burden of malaria parasites. Pre-erythroytic stage is clinical silence, but is the promising stage to design subunit vaccine against malaria parasites. Such a vaccine would prevent the development of clinical symptoms and the transmission of malaria.Irradiation-attenuated sporozoite is still the only effective vaccine that could induce sterile protective immunity. However, limitation of purified sporozoites and difficulty in controlling attenuation degree of sporozoites by irradiation hampered its use in practices. However, understanding the mechanism of long-term sterile protection induced by the irradiation -attenuated sporozoites would help the design of new effective subunit vaccine against malaria parasites. In the present study, we investigated whether irradiation -attenuated sporozoites could induce sterile protective immunity, and possible mechanisms responsible for the induced protective immunity.1. Establishment of the Real-time PCR for detecting pre-erythrocytic Plasmodium. P.y. BY265 18SrRNA was amplified with primers designed according to their conservative region of Plasmodium 18S rRNA. A amplified fragment with 839bp in length was routinely cloned into pMD18T vector, and sequencing. Then, the specific primer and TaqMan probe were designed according to the sequence of P.y. BY265 18S rRNA. With mouse GAPDH as control, the load of the Plasmodium liver stage was indicated as P.y. BY265 18S rRNA abundance. Our data showed that the method could discriminate different liver parasite load and detect liver parasite load in mice challenged with as low as 50 infectious sporozoites.2. Investigating sterile protective immunity induced by irradiated-attenuation sporozoites and its effectors.1) To determine the optimal irradiation dose of the attenuated sporozoites:After irradiation attenuated with 60Co in dose of 8000rd and 10000rd for 80min at room temperature, respectively, each of 10,000 attenuated sporozoites were injected into mice by vena caudalis. Liver Plasmodium burden was detected with the established Real-time PCR at 42 hours post-infection, and the parasitemia was also monitored from 4d to 14d post-infection. Althoug no blood stage was detected in both two groups, Real-time PCR showed that plasmodium 18SrRNA could be detected in mice inoculated with sporozoites irradiated at dose of 8,000rd, but not in those injected with 10,000rd-irradiated sporozoites. So 8,000rd was selected to irradiate sporozoites .2) Sterile protective immunity induced by irradiated-attenuation sporozoites:14d after the last immune, mice immunized with or without iiradiated-attenuation sporozoites were attacked by 1000 sporozoites through vena caudalis. Although the parasitemia in the non-immune mice reached for 10% at 5d post challenge, no blood stage was observed in immunized mice till 14d. It showed that irradiation attenuated sporozoites could induce mice to produce sterile protective immunity .3) Irradiated-attenuation sporozoites induce mice to produce sporozoite-specific antibody and activate CSP-specific CD8+ T cellAt 30d after last immunization, the serum of tow groups mice were collected, the anti-sporozoites antibody titer in serum were detected by indirect immuno- fluorescence assay. Results showed that the anti-sporozoites antibody titer in serum were 1:200 and 1:400 in two immunized mice, respectively, but no signal was detected in the serum of no-immunized mice; At 45d after the last immune, IFN-γsecreted by CSP-specific CD8+ T cells in liver was measured after stimulation of CD8+ T specific epitope from CSP, SYVPSAEQ, for 18h. We found that IFN-γsecreted by the liver leukomonocyte of immunizeded mice secrete a large near to positive control, negative control could not secrete IFN-γ.In conclusion, we determined the optimal dose for sporozoite to be irradiated attenuation, and found immunization of attenuation P.y BY265 sporozoite could induce the mice to produce sterile protective immunity against the challenge of wild-type sporozoites, which was associated with sporozoite-specific antibody and CSP-specific CD8+ T cell induced by Irradiated-attenuation sporozoites.
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