Study Of Epidemic Characte Ristics Of Malaria Along China-Myanmar Borde R And The Novel TBV Candidate Antigen Pb61

Objective:According to the World Malaria Report 2018,219 million people around the world were still infected and 435000 died from malaria in 2017.The World Health Assembly approved the Global Malaria Technology Strategy for 2016-2030,which calls for a global reduction of malaria morbidity and morta lity at least 90%by2030.This ambitious plan should eliminate all types of malaria infections,including severe and complicated,mild and uncomplicated as well as asymptomatic infections.Although malaria control stratergies are effective at present,the emergence of insecticides and antimalarial drug resistance and the recessive transmission of asymptomatic infection carriers pose a great threat to malaria control and elimination.Faced with these challenges,vaccines are undoubtedly the best strategy to achieve the malaria eradication ambitious plan.Compared with other vaccine candidate antigens,transmission blocking vaccines?TBVs?candidate antigens were expressed on the membrane surface of Plasmodium parasites in the sexual stage,and were not subjected to the immune pressure of human host,and the antigenic polymorphism was lower.Therefore,in endemic areas,TBV is the most effective strategy to prevent the transmission of malaria parasite from mosquito vector to human and eventually to eradicate malaria.However,the development of TBVs is still slow,especially in Plasmodium vivax.Experimental evidence showed that not all homologous genes can play an important role in the corresponding malaria species.This study is based on the high prevalence of asymptomatic malaria infection,through bioinformatics analysis,the candidate antigen of sexual stage of malaria parasite with unknown function was selected,and its molecular structure,expression stage,transmission blocking activity and biological function were further determined.These were all to develop safer and more efficient TBV for the total elimination of all types of malaria.Laiza town along China-Myanmar border as the survey site,and three cross-sectional surveys on healthy population were conducted respectively in early season,peak season and late season.Molecular detection was used to determine the prevalence of asymptomatic malaria infection and gametocyte carriers.Next,we used Plasmodium berghei for primary screening.By bioinformatics analysis,PBANKA₁45910?Named Pb61?was selected,which was the membrane surface protein and expressed sexual stages,and its dominant epitope region was intercepted.The antigenicity and immunogenicity of the selected protein were evaluated by using the serum antibody after direct immunization of the recombinant protein.The localization and expression characteristics of Pb61 in Plasmodium berghei were determined by immunological experiments.The transmission blocking effect of Pb61 was verified by in vivo and in vitro assays.The?Pb61 parasites were obtained by double cross homologous recombination,and the phenotypic analysis and biological function of?Pb61 were analyzed in detail.The effect of Pb61 gene on the whole life cycle of Plasmodium was clarified,which provided theoretical basis for Pb61 to be a candidate target for transmission blocking vaccine or antimalarial drugs.Methods:1.Sample size is estimated by formula method.The formula for calculating the sample size of random sampling is:n=??²?????1-??/?²,and the malaria prevalence rate???at the border is 20%.The statistical value of?as 95%confidence interval?CI?is 1.96 and the error???is 2%.2.Identification of asymptomatic malaria infection by nRT-PCR.Nested polymerase chain reaction?nRT-PCR?is based on the18s rRNA gene described previously.The detected human malaria species include P.falciparum,P.vivax,P.ovale and P.malariae.3.qRT-PCR was used to identify the gametocytes carriers.The specific primers of Pvs25,which were specifically expressed on the surface of gametocytes,were used to further identify the gametocyte carriers of the P.vivax samples through TaqMan probe qRT-PCR.4.Bioinformatics analysis.The sequence of PBANKA₁45910?Pb61?gene with unknown function was obtained from PlasmoDB website.Through bioinformatics analysis software,the homology analysis was carried out,and the domain,signal peptide,transmembrane region and antigen epitope of pb61 were predicted.5.Expression and purification of rPb61.The target fragment of Plasmodium berghei gDNA was amplified and the pET32a-pb61 expression vector was obtained by vector constructio n system.The strains of identified pET-32a?+?-pb61 were expanded and induced to express rPb61by IPTG.The recombinant rpb61 protein was purified by affinity chromatography and the protein size was determined by SDS-PAGE and Western blot.6.Detection of serum specific antibody against rPb61.rPb61 was mixed with Freund's adjuvant and then injected subcutaneously to female BALB/c mices.PBS dissolved in rPb61 as control.Serum was collected on day 14,28,42 after the first immunization.Using the parasite protein of Plasmodium berghei as antigen,serum antibody titers and immunogenicity of Pb61 were verified by ELISA and Western blot.7.Transcription level of Pb61 mRNA was detected by RT-PCR.Total RNA were extracted from purified schizonts,gametocytes and ookinete according to the instructions of tissue RNA extraction kit.Total RNA obtained using PrimeScript^T M M RT reagent Kit with gDNA Eraser to obtain cDNA.Primers were designed to detect the transcriptional level of Pb61 mRNA in the three stages of Plasmodium berghei by real-time PCR.8.Indirect immunofluorescence assay.The parasites of each stage was treated with 0.1%triton x-100 and untreated,and then incubated with rPb61 antiserum and Alexa Fluor488 labeled anti-mouse IgG?PBS antiserum as negative control?.After DAPI staining,the parasites were sealed with anti-quenching tablet?ProLong?Gold antifade reagent?.The results were observed by fluorescence microscope.9.The acquisition of Pb61-HA tag parasite strain.After expanded culture,Pb61-HA tag plasmids were extracted by plasmid extraction kit and identified by PCR.The linearized Pb61-HA tag plasmid was electrotransfected into the purified schizont and was injected into mouse by tail vein to culture in vivo.The high purity of Pb61-HA tag parasite was obtained by monoclonal method after screening with pyrimethamine,and the successful acquisition of Pb61-HA tag parasite was confirmed by PCR and Western blot.10.The expression characteristics of Pb61 were detected by Western blot.Purified wild type and Pb61-HA tag schizont,gametocyte and ookinete protein were separated by SDS-PAGE.The expression stage of Pb61-HA tag was determined by the reaction of anti-HA tag rabbit antibody with PVDF membrane printed with each phase antigen.The wild type and Pb61-HA nucleus,organelle,cytoplasm and plasma membrane protein were separated by SDS-PAGE.The expression characteristics of Pb61 were also determined by the reaction of anti-HA tag rabbit antibody with PVDF membrane with each antigen.11.Evaluation of the transmission blocking effect of Pb61 in vivo and in vitro.In vitro,blood of mice mixed with ookinete culture medium 1:4?mouse antiserum containing diluted rPb61/PBS:1:5/1:10/1:50?.The exflagellation was observed under optical microscope after culture.The blood of mice mixed with medium 1:9?containing the same diluted each antiserum?.After culture,the number of the ookinetes in the two groups was counted.In vivo,mice immunized by rPb61 and PBS were injected intraperitoneally with Plasmodium berghei.On the 4th day after infection,mice were fed starved female Anopheles.Post 10 days of direct feeding,female Anopheles mosquitoes were dissected and the number of oocysts was compared.12.?Pb61 parasite obtainment.The?pb61 plasmid was extracted by plasmid extraction kit and identified by PCR.The linearized?pb61 plasmid was electrotransfected into the purified schizont and was injected into mouse tail vein to culture in vivo.The highly purified?pb61parasite was obtained by monoclonal method after screening with pyrimethamine,and the successful acquisition of?pb61 parasite was confirmed by PCR,RT-PCR and Western blot.13.Comparison of gene function between?pb61 and wild WT parasite.?pb61 and wild type parasites were injected intraperitoneally into 5 BALB/c female mice in each group.From the 3rd day after infection,the parasitemia,the gametocytemia and ratio of female and male gametocytes were counted and the survival of mice was recorded daily.On the second day after infection,exflagellation was observed dynamically for 7 days.On the 3rd day after infection,the number of ookinete cultured in vitro was counted,and the number of ookinetes and oocysts in vivo were compared by direct membrane feeding assay.14.Statistical analysis.Graphpad prism 6.0 and SPSS statistics 19.0 were used to process and analyze the data.P<0.05 means statistically significant.Results:1.Sample size.According to the formula,the sample size is at least 1537cases,while the actual sample size is 1680 cases,including in the early season?n=516?,the peak?n=578?and the late season?n=586?.2.Asymptomatic P.vivax infection with gametocyte showed high prevalence along China-Myanmar border.The results of nRT-PCR for 18s rRNA showed that most malaria infections were asymptomatic submicroscopic infections,with 23.4%prevalence.P.vivax was the main asymptomatic infection species,accounting for 89.6%of all infection species.Of the 326 P.vivax samples,181 cases were confirmed to be gametocyte positive by qRT-PCR,and with 55.5%prevalence.3.Successfully cloning,expression and purification of rPb61.According to the bioinformatics analysis,the expression region of Pb61 was selected?284aa-384aa?.We successfully constructed the pet32a-pb61plasmid and transformed it into the E.coli expressing strain BL-21.The recombinant protein was purified by His-tag-Ni-NH₃-triacetic acid agarose column.The results of SDS-PAGE and Western blot showed that the recombinant protein with a high purity of about 31kDa was obtained.4.High titer of rPb61 immuno-specific serum antibody was successfully obtained.The results of ELISA showed that the level of antibody?OD490 nm?after three times of rPb61 immunization was significantly higher than that of the control group.The titer of anti-serum obtained from rPb61immunized mice reached 1:6400.Western blot showed that the rPb61 immunized antiserum could specifically recognize the bands of about 61 k Da,which indicated that Pb61 had good antigenicity.5.mRNA of Pb61 gene was transcribed mainly in sexual stage.RT-PCR results showed that the transcription level of mRNA of Pb61gene in gametocyte and ookinete stage was much higher than that in schizont stage when schizont as reference.The results showed that Pb61 mRNA mainly transcripted during sexual stage.6.Pb61-HA tag parasite was successfully obtained.We obtained the drug-resistant Pb61-HA tag parasites after the selection of pyrimidine.The results of PCR and Western blot showed that the Plasmodium berghei with HA tag was successfully obtained at the C'terminal of Pb61.7.Pb61 was mainly expressed on the membrane surface of gametocyte and ookinete.The IFA results of 0.1%tritonx-100permeation and non-permeation showed that Pb61 was localized at all stages of the Plasmodium,but mainly on the cytoplasm and membrane surface in gametocyte stage and on the membrane surface at the late stage of fertilization.Western blot analysis showed that Pb61 also was mainly expressed in gametocyte and ookinete stage,and the membrane surface of the gametocyte.8.Antiserum of rPb61 has obvious transmission blocking activity.The results of in vitro experiments showed that the antiserum of rPb61 could inhibit the male gamete formation and decrease the number of ookinete after 24 hours,and the inhibitory effect depended on the dosage of antibody.The results of three independent experiments in vivo showed that there was no significant decrease in mosquito infection rate compared with the control group,but the number of oocysts decreased by 54.53%,56.26%and 36.85%,respectively.The difference was statistically significant.9.?Pb61 parasites were successfully obtained.The?pb61 parasite was screened by pyrimethamine.After monoclonal,the results of PCR,RT-PCR and Western blot all showed that the parasite of?pb61had been successfully obtained.10.The sexual stage development of?pb61 was blocked.There was no significant difference in parasiyemia and survival rate of mice between?pb61 and WT,the number of gametocyte and the ratio of male and female gametocytes had no significant difference.However,the formation of male gamete was totally inhibited,and ookinete was not formed in vivo and in vitro,and the oocyst was not found in the midgut of mosquitoe.Conclusion:1.Asymptomatic infections serve as important reservoirs of continued malaria transmission.2.Pb61 is mainly expressed on the membrane surface of sexual stages.3.Antiserum of rPb61 has good immunogenicity and significant transmission blocking effect.4.Pb61 plays a key role in the formation of male gamete.