The Construction And Antitumor Effect Of Attenuated Plamodium Berghei With NT1 Gene Knockout

Maiaria is one of the three most aevasiating diseases world-wiae,caused by protozoan parasites of the Plasmodium which is transmitted by female Anopheles mosquitoes.About 3.2 billion people are under the threat of malaria infection,and 95%deaths are caused by Plasmodium falciparum.Lung cancer is one of the most common malignant tumor in the world,whose morbidity and mortality are the highest amont the tumors.The traditional surgery,chemotherapy and radiation radiotherapy encountered the bottlenecks for their respective limitations,for examples,lack of specifity immune response for tumor.In the previous study of our lab,malaria parasite infection significantly suppressed Lewis lung cancer growth and prolonged survival time of tumor-bearing mice via induction of innate immunity and antigen-specific antitumor immune response in a mouse model.However,the wild-type malaria parasite had some virulence,we aimed to attenuate for improving the safety in lung cancer treatment using malaria parasite.Thus,it is important to develop effective malaria vaccines for preventing people from malaria infection and eliminating malaria.The live attenuated vaccine has been recognized as an efficient strategy.Plasmodium spp.are unable to synthesize purine rings de novo,while they uptake the purine nucleosides and purine nucleobases by the nucleoside transporters 1(NT1)from their hosts to support their proliferation.And NT1 is a key enzyme in the purine synthesis pathway in Plasmodium spp.Plasmodium berghei ANKA(P.b ANKA)is a lethal murine malaria parasite strain in C57BL/6 mouse model.Therefore,P.b ANKA infection in C57BL/6 is the best experimental model for attenuated malaria vaccine development.In this research,the vector of pL0018-NTl that containing the report gene of GFP and the resistant gene of tgdhfr/ts was constructed and electroporated into wild type P.b ANKA for double-crossover homologous recombination,and the genetically modified parasite was selected using pyrimethamine.We got two clones(labeled as 8#and 9#)that had GFP expression and the specific integration after first round of monoclonalization.Selectively,the clone 8#was used to continue the second round of monoclonalization and the stable NT1 gene knockout strain of P.b ANKA-ANT1-8C1 was obtained.The parasitemia of the P.b ANKA-ANTI parasites infected C57BL/6 mice could be as high as 85%and the infection cycle could be as long as 80 days.Five of the six mice infected with P.b ANKA-ANT1 parasites were still alive during this long infection cycle.But the mice infected with wild-type P.b ANKA were dead quickly.This means that the lifetime of the mice infected with the NT1 gene knock-out parasite were significantly longer than those infected with the wild-type parasite.Interestingly,we found that the mice infected with P.b ANKA-ANT1 strain experienced a significant decrease in the number of gametocytes in their peripheral blood during 38 days after infection.The gametophyte number of P.b ANKA-ANTI strain was up to 48 per 100 white blood cells(WBCs)in a field of view under microscope(100×),and was fluctuated between 0 and 25 during the initial 2-to 18-infection days,which was far lower of wild-type P.b ANKA.The results indicated that NT1 gene knockout is useful to lower the sexual reproduction efficiency of malaria parasites in female Anopheles mosquitoes.This strategy may benefit on blocking malaria transmission,and may be much safer than those strategies using drug or irradiation to attenuate.We also found before that the longer of parasite infection cycle,the better inhibition of tumor growth.The infection with P.b ANKA-ANTI strain had a longer blood-stage(80 days)course than that(30 days)with Plasmodium yoelii.In fact,tumor-bearing C57BL/6 mice with P.b ANKA-ANT1 parasite infection more significantly inhibited tumor growth and had a longer survival time(5 more days)than that with the wild type P.b ANKA infection.To mice,in other words,this strategy changed a lethal parasite into a nonlethal parasite.The longer infection cycle and the obvious reduction of gametocytes formation in the peripheral blood makes the P.b ANKA-ANT1 strain to be a more effective and safe live attenuated whole-parasite vaccine candidate.The P.b ANKA-ANT1 strain also can be used as a vector to produce lung cancer vaccines by expressing the specific lung cancer antigens.This research will provide a novel method and idea for biological immunotherapy of tumors.At the same time,the attenuated P.b ANKA-ANTI strain used as a malaria vaccine for protecting the people health and eliminating malaria.