| Objective:Malaria is a vector-borne disease caused by protozoan plasmodium parasites in the genus Plasmodium,and is spread through female Anopheles mosquitoes.It remains one of the devastating infectious diseases and carries a serious social and economic burden in developing countries.In the past decade,significant progress in malaria control has led to the renewed interest in malaria elimination.Yet,for the realization of this goal,malaria-endemic countries face a number of technical challenges.Given that the malaria life cycle involves both humans and mosquitoes,integrated control measures targeting stages in both hosts are desired.Current malaria management relies heavily on effective chemotherapy to treat infected patients and on vector control such as insecticide treated bednets and indoor residual sprays.However,these measures are compromised by the emergence of drug-resistant malaria parasites and insecticide resistance mosquitoes.Whereas vaccine is considered a cost-effective tool that is highly significant for eliminating malaria,the leading vaccine RTS,S/AS01,a subunit vaccine targeting the circumsporozoite protein,has only proven to be partially effective.Current vaccine development efforts explore antigens of multiple stages of the malaria parasite life cycle,including transmission-blocking（TB）vaccine（TBV）that targets sexual development.TBVs aim to induce immune responses that interfere with parasite transmission to the mosquito and,consequently,block disease spread between humans.Despite their importance,only two candidates Pfs25 and pfs230 have reached the preclinical phase in Malaria Vaccine Initiative’s development pipeline（www.malariavaccine.org）.TBV candidates can be antigens expressed on gametocytes and gametes（pre-fertilization）,or on zygotes and ookinetes（post-fertilization）,or even antigens expressed by the mosquito midgut.The well-documented presence of naturally acquired TB immunity in human populations living in malaria-endemic areas is probably resulted from the antigens released from dead gametocytes,and thus TBVs targeting the pre-fertilization antigens will have the advantage of boosting natural immunity.Among the leading TBV candidates P25/28,P48/45,P230,and HAP2,P25/28 is expressed from female gamete till early oocyst development.Antisera against Pfs25 have the ability to completely inhibit oocyst development in mosquitoes.Pfs230 and Pfs48/45 are localized on the outer membrane of gametocytes and play roles in gamete-gamete recognition and fertilization.Pfs230 and Pfs48/45antigen-specific IgGs show the ability to prevent oocyst formation,probably through lysis of gametes in a complement-dependent manner.HAP2,also referred to as GCS1（generative cell specific 1）,is expressed on male gametocytes and microgametes,and is indispensable for gamete fusion.Whereas the anti-Plasmodium berghei HAP2serum showed no influence on male gamete formation,it significantly interfered with ookinete and oocyst development.The mosquito midgut ligands carboxypeptidase B and alanyl aminopeptidase N1（APN1）are TBV antigens expressed by the mosquito midgut.Antibodies against Anopheles gambiae APN1 possess strong TB activity against P.falciparum in different mosquito species.Although TBVs based on Pfs25and Pvs25 have been evaluated in phase I clinical trials,TBV development still encounters a number of difficulties.For the lead TBV candidates,it often requires the expression of native proteins in the correct conformation in order to induce effective TBA.Even so,most TBV candidates could not induce complete TBA,and a successful TBV will probably need to combine antigens expressed in both pre-and post-fertilization stages in order to achieve complete blocking of transmission.With the limited number of TBV candidate antigens currently available,further efforts in TBV antigen discovery are required.With our efforts in TBV antigen discovery and functional studies of genes during sexual development of the malaria parasites,we identified a sexual stage gene that is highly conserved in Plasmodium.Characterization of this gene（PBANKA060330）in the rodent parasite P.berghei,designated as Pbg37,revealed its expression in both pre-and post-fertilization stages.While deletion of this gene affected of male gametocytogenesis and functions,immunization with the recombinant Pbg37 protein（rPbg37）resulted in a modest level of transmission-blocking activity in direct mosquito feeding experiments.METHODS:1)Mice,parasites and mosquitoes.BALB/c mice aged six-to eight-weeks were bought from Beijing Animal Institute.The P.berghei ANKA strain was maintained by serial passages as described previously.Mouse infection was initiated by intraperitoneal injection of 1×107 P.berghei-infected red blood cells（RBCs）.Anopheles stephensi mosquitoes（Hor strain）were maintained in an insectary at 25°C and 50–80%humidity.All animal experiments were approved by the animal ethics committee of China Medical University.2)Expression and purification of recombinant protein.For the expression of recombinant Pbg37（rPbg37）,total RNA was extracted from purified gametocytes and RT-PCR was performed to amplify the pbg37 fragment encoding amino acids（26-88）with primers pbg37-F and pbg37-R.The PCR product was digested with Not I and Bam HI and cloned into p ET32a（+）.Expression of the rPbg37 in Escherichia coli Rosetta-gami B（DE3）cells was induced with 1 mM IPTG（isopropyl-D-1-thiogalactoside）（Sigma）at 20°C for 8 h and rPbg37was purified on a Ni-NTA agarose column（Novagen）.His-tag protein expressed from the empty vector pET32a（+）was purified similarly and used as a negative control for immunization.Purified recombinant proteins were dialyzed extensively in phosphate buffered saline（PBS,p H 7.4）at 4°C overnight and analyzed on a 10%SDS-PAGE gel.3)Immunization of BALB/c mice.BALB/c mice（n=5）were immunized with rPbg37（50μg each）emulsified in complete Freund’s adjuvant（Sigma）via subcutaneous injection.Two booster immunizations with rPbg37（25μg/mouse）were done at two weeks interval.The same immunization procedure was followed with another group of mice（n=5）using the negative control protein.Serum was collected and pooled together for each group of mice on the day before each immunization and at 14 days after the final immunization.Enzyme-linked immunosorbent assay（ELISA）was used to assess the antibody titers in the rPbg37 immunization group as previously described.To estimate the end point titer of antiserum from the rPbg37-immunized group and the control group,serum was serially diluted in 1%bovine serum albumin（BSA）in phosphate buffered saline（PBS,pH 7.0）from 1:2000 to 1:128000.The end point is defined as the highest dilution of the antiserum when the optical density（OD）value at 490 nm was above the average of the control antiserum+3×standard deviation.4)Western blot and indirect immunofluorescence assay（IFA）.To determine the expression of Pbg37 protein in different development stages of the parasite,P.berghei schizonts,gametocytes,and ookinetes were purified on Nycodenz gradients essentially as previously described.Proteins from purified parasites were extracted with 2%SDS containing protease inhibitors.Equal amounts of parasite proteins（10μg）were separated in 10%a SDS-PAGE gel under reducing conditions,and transferred to a PVDF membrane（Bio-Rad）.The membrane was blocked with5%non-fat milk in TBS-T（Tris-buffered saline with 0.1%Tween 20）at room temperature for 2 h and probed with the mouse anti-rPbg37 antisera（1:200）.After three washes with TBS-T,the membrane was incubated with HRP-conjugated anti-mouse antibody.Protein bands were visualized by chemiluminescence using a Pierce ECL Western Blots Kit.The localization of Pbg37 in parasites was detected by IFA.Parasites were fixed in a tube with 4%paraformaldehyde for 30 min at room temperature then washed once with PBS.The fixed cells were either permeabilized with 0.1%Triton X-100 in PBS for 10 min and then blocked with 3%BSA/PBS for60 min or blocked without permeabilization.Following another PBS wash,mouse anti-rPbg37 antisera（diluted to 1:200 in 3%BSA/PBS）were added and incubated at37°C for 1 h.After removing excessive primary antibodies with PBS,FITC-conjugated goat anti-mouse antibody（Invitrogen）diluted to 1:500 in 3%BSA/PBS was added and incubated for 1 h.Parasite nuclei were counterstained with10μl DAPI（Sigma）.Slides were mounted with ProLong?Gold Antifade Reagent（Invitrogen）and observed under Olympus BX53 microscope.Pbs21 monoclonal antibody was used to show surface staining of ookinetes.Control sera from mice immunized with His-tag protein were used as negative controls.5)Generation of transgenic parasites.A double-crossover homologous recombination strategy was used to generate a parasite line with pbg37 gene deletion（Δpbg37）.The pbg37 5′and3′flanking regions were amplified using primer pairs 5UTR-F×5UTR-R and3UTR-F×3UTR-R,respectively,and cloned into the vector to flank the human dhfr expression cassette.Tenμg of the plasmid was linearized by Hind III and Xho I digestion,and electroporated into purified P.berghei schizonts using Nucleofactor?.After transfection,parasites were mixed with 50μl of complete culture medium and inoculated into mice.Twenty four hours later,parasites were selected with pyrimethamine（70μg/ml）via the drinking water for mice.Parasite genomic DNA was extracted from infected blood to confirm pbg37 gene deletion using integration-specific diagnostic PCR with primers P1×P2,P1×P3,P4×P5.6)Phenotype analysis of theΔpbg37 parasites.To study the effect of pbg37 deletion on parasite development,five mice were injected intraperitoneally with 1×106Δpbg37infected RBCs（clone KO1 and KO2）and another five injected with same number of wild type P.berghei-infected RBCs.Parasitemia and gametocytemia were monitored daily by microscopic examination of Giemsa-stained thin blood films.In each mouse,a total of 100 gametocytes were differentiated into male and female to determine the gametocyte sex ratio.The proportion of mature gametocytes in the total gametocyte population was also estimated in a total of 100 gametocytes.Exflagellation of male gametocytes and formation of ookinetes were monitored using in vitro cultures.Briefly,infected blood containing equal number of mature male gametocytes was mixed with the ookinete culture medium and incubated at 25°C for 15 min to induce gamete formation.The number of exflagellation centers in each 100 x field was counted under a phase contrast microscope.A total of twenty fields were counted per mouse.To observe ookinete formation,blood samples were incubated at 19°C for 24h and ookinetes were monitored by IFA using the monoclonal antibody against Pbs21.Infectivity of the parasites to mosquitoes was evaluated in a direct mosquito feeding experiment.On day 3 p.i.when asexual parasitemia had reached 5-7%,4 day-old starved An.stephensi mosquitoes（50 per mouse）were allowed to feed on the infected mice for 1 h.Fully engorged mosquitoes were kept for 12 days and approximately 30mosquitoes of each group were dissected.Midguts were stained with 0.5%mercurochrome for 10 min to determine the oocyst number per positive midgut and mosquito infection rate（the number of infected mosquitoes/the number of total mosquitoes）.7)Quantification of TB activity.The TB potential of pbg37 was assessed using an in vitro ookinete formation assay and direct mosquito feeding assay.For the ookinete formation assay,mice were infected with intraperitoneal injection of1×106 P.berghei-infected RBCs after treatment with phenylhydrazine.At 3 days p.i.,anti-rPbg37 sera or negative control sera were diluted at 1:5,1:10 and 1:50 with the ookinete culture medium,and mixed with 10μl of infected mouse blood in a total volume of 50μl.Exflagellation of male gametocytes and ookinete formation were quantified as described above.For mosquito feeding experiments,mice were immunized with rPbg37 or the negative control protein as described above.On day 14after the final immunization,mice were infected with 1×106 parasites.Three days after infection,mice were fed on starved female A.stephensi（50 mosquitoes/mouse）.Oocyst density and mosquito infection rate were determined as described above.8)Statistical analyses.The analyses were carried out using SPSS software,version 17.0（SPSS Inc.,USA）.Student’s t-test was used for statistical comparison between groups of asexual parasitemia,gametocytes,sex ratio,mature gametocytemia,exflagellation and ookinete numbers.Mann Whitney U test was employed to analyze oocyst density（oocysts number/midgut）,while Chi-square test was used to analyze infection prevalence.All data were presented as mean±standard error of the mean（SEM）.P<0.05 was considered statistically significant.Results:1)Pbg37 is a highly conserved protein expressed in Plasmodium sexual stages.Using a bioinformatic scheme defined earlier,we searched PlasmoDB for（1）“conserved”proteins present in Plasmodium spp.,（2）the presence of putative signal peptides,（3）the presence of one or more predicted transmembrane region,and（4）abundant expression in gametocyte stages.This led to the identification of 13 genes with these characteristics and transcript abundance in the top 97%percentile of the RNA-seq data in the P.berghei gametocyte transcriptome.Among them,we selected PBANKA060330 in P.berghei for detailed functional studies.This gene encodes349 amino acids with a predicted molecular weight of 40 kDa.The predicted protein contains a putative signal peptide（amino acids 1-23）,seven transmembrane regions,and one low complexity region.Whereas BLAST analysis did not identify homologous sequences to this gene in organisms outside of Plasmodium,this gene is highly conserved among Plasmodium species as shown from the results of multiple sequence alignments.Based on the predominant expression of this gene in the gametocyte stage of P.berghei from RNA-seq analysis and the predicted mature protein size of37 kDa after the cleavage of the signal peptide,we named this protein as Pbg37.2)Recombinant Pbg37 induces high IgG titers in immunized mice.Because Pbg37 is predicted to have a signal peptide and the multiple transmembrane domains,we selected a small 63-amino acid fragment between the signal peptide and the first transmembrane domain（amino acids 26-88）for protein expression in bacteria.Since Pbg37 is predicted to contain 12 introns,RT-PCR was performed using P.berghei gametocyte mRNA and sequencing of the PCR product confirmed the gene model.His-tagged rPbg37 protein was purified by Ni-NTA affinity chromatography to almost homogeneity as shown in SDS-PAGE gel and had a molecular weight of approximately 27.9 kDa（contains the His-tag）,which agrees with its predicted size.Mice were immunized with the rPbg37 protein to produce polyclonal antibodies.Antibody titers assessed by ELISA showed that the second booster immunization induced a significantly higher titer than control（P<0.0001）,reaching an end point titer of 1:64000 after final immunization.This result showed that under the immunization scheme used,rPbg37 protein was highly immunogenic and induced specific antibodies in mice.3)Pbg37 is expressed in sexual stages.To determine the expression of Pbg37 protein during parasite development,equal amounts of lysates from purified schizonts,gametocytes,zygotes and ookinetes were separated by SDS-PAGE and immunoblotted with the polyclonal mouse anti-rPbg37 sera.Consistent with predominant expression of the Pbg37 mRNA in sexual stages,a37kDa band was recognized by the anti-rPbg37 sera in the lysates of gametocytes,zygotes and ookinetes,whereas no specific protein bands were detected in the lysate of schizonts.The size of the detected protein band agreed well with the predicted molecular weight of Pbg37 after cleavage of the putative signal peptide.As compared with the similar amounts of HSP70 protein detected in all parasite stages tested,the Pbg37 band in ookinetes appeared slightly lighter,suggesting that less Pbg37 was present in ookinetes.In comparison,control sera showed no specific reactions with the parasite lysates.Given that the predicted protein contains a signal peptide and transmembrane domains,we wanted to determine the localizations of the Pbg37protein during sexual development.Consistent with the Western blot,IFA did not detect Pbg37 expression in schizonts.Interestingly,sex difference in Pbg37expression was noted,and Pbg37 appeared more abundant on male than female gametocytes.In the exflagellating male gamete,Pbg37 was mostly associated with the residual body,while speckled fluorescence was also observed on the microgamete.In comparison,weaker fluorescence was observed on the macrogamete and the fluorescence had a spotty appearance.Whereas strong surface staining of zygote was observed,the signal was gradually reduced during subsequent transformation and only residual fluorescence was observed on the retort and ookinete stages.It is noteworthy that the fluorescence was observed at gametes,zygote,retort and ookinete with membrane permeabilization,whereas the fluorescence was observed at gametocytes,gametes,zygote,retort and ookinete without membrane permeabilization,indicating that at least the N-terminal domain of Pbg37 was localized on the outer membrane of these sexual stages.Control sera showed undetectable levels of staining in these developmental stages.4)Pbg37 is important for sexual stage development especially male gametocytes.To determine the function of Pbg37 during P.berghei development,a pbg37 knockout line（Δpbg37）was generated using a double crossover strategy.After transfection,parasites were selected with pyrimethamine,and pbg37 deletion was verified by integration-specific PCR.Phenotypic analysis of theΔpbg37 parasites（clone KO1 and KO2）were performed in parallel with the wild-type parasite strain.Consistent with the lack of pbg37 expression in asexual erythrocytic stages,theΔpbg37 lines and the wild-type control showed no significant difference in daily asexual parasitemia.However,theΔpbg37 lines showed significantly lower daily gametocyte densities than the wild-type parasite（P<0.0001）,though the dynamics of gametocytogenesis was similar.On day 3 p.i.when gametocytemia reached peak levels,there was a60%reduction in gametocyte density in theΔpbg37 line（P<0.0001）.In addition,deletion of pbg37 had a substantial impact on the sex ratio with theΔpbg37 lines having a significantly higher female/male ratio（6:1）than the wild-type parasite（3:1）（P<0.0001）.Whereas the gross morphologies of the male and female gametocytes in Giemsa-stained thin smears in theΔpbg37 lines were similar to those in wild-type parasites,deletion of pbg37 influenced the development and maturation of microgametocytes.At 3 days p.i.,theΔpbg37 parasites showed a significantly lower proportion of mature male gametocytes than the wild-type group（P=0.007）,whereas the proportions of female gametocytes in theΔpbg37 lines and the wild type were not significantly different（P>0.05）.Furthermore,even though the mature microgametocytes appeared morphologically normal,they exhibited defects in exflagellation.When the same numbers of mature male gametocytes were compared between theΔpbg37 lines and the wild type in an in vitro exflagellation assay,theΔpbg37 lines had a36%decrease in the number of exflagellation centers per field as compared to that in the wild-type parasites（P<0.0001）.Altogether,these phenotypic analyses showed more severe developmental defects in male gametocytogenesis in theΔpbg37 lines.We next determined the consequence of pbg37 deletion on fertilization and subsequent development using an in vitro assay.Even when the same number of mature male gametocytes was used in the in vitro ookinete culture,the pbg37 deletion lines showed a75%decrease in the ookinete number（P<0.0001）,while the numbers of zygotes and retorts at the end of culture were similar betweenΔpbg37lines and the wild type.Mosquitoes fed on mice infected with theΔpbg37 lines showed a dramatic reduction in terms of infection rate and oocyst density compared to those fed on mice infected with the wild-type parasites.The prevalence of mosquito infection in theΔpbg37 group（68.2%）was much lower than in the wild type group（93.1%）（P<0.0001）.In addition,there was a 91.4%average reduction in the mean oocyst number/midgut in mosquitoes fed on mice infected with theΔpbg37 parasites（12.2）as compared to that in wild type group（140.9）（P<0.0001）.5)Anti-rPbg37antibody has modest TB activity.The surface expression of Pbg37 in sexual stages prompted us to evaluate the TB potential of Pbg37 using both in vitro ookinete formation and direct mosquito feeding assays.Incubation of the microgametocytes with the anti-Pbg37 antisera at 1:5,1:10 and 1:50 dilutions led to 70%（P<0.0001）,65%（P<0.0001）and 60%（P<0.0001）decrease of exflagellating males,respectively.In an in vitro ookinete formation assay,the anti-Pbg37 antisera at 1:5,1:10 and 1:50 dilutions reduced the ookinete number by 71%（P<0.0001）,58%（P<0.0001）and 52%（P<0.0001）,respectively.To test the TB potential of Pbg37 in vivo,mice were immunized with the rPbg37 using our established immunization scheme and then direct mosquito feeding with An.stephensi was performed on day 14 after the final immunization.Whereas rPbg37 immunization with only resulted in a slight（9.23%）reduction in the prevalence of infected mosquitoes（P=0.094）,it resulted in a 49.1%decrease of the oocyst density（P<0.0001）.Conclution:1)Pbg37 encoding a protein of 37 kDa with signal peptide and multiple transmembrane domains.2)Pbg37 is preferentially expressed in gametocytes.3)Pbg37 demonstrated a more substantial influence on the development and maturation of male gametocytes,and as a result,theΔpbg37 line exhibited a higher female/male ratio,fewer mature male gametocytes,and a lower number of exflagellation centers even from the same number of mature male gametocytes.4)Antibodies against rPbg37 showed obvious transmission-blocking activities. |
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