Evaluation Of Pbg37-PSOP25 And PSOP25-PSOP26 As Bivalent Transmission-blocking Vaccines In Rodent Malaria

Objective:Malaria is one of the most serious vector-borne diseases that still threaten global public health.Due to the complex feature of the transmission of malaria parasites between human and mosquitoes,there are still many difficulties in the treatment and prevention of malaria.According to the World Malaria Report,approximately 228million cases of malaria infections and 400,000 cases of deaths were reported globally in 2018.Vaccine,as one of the important methods in malaria prevention and control,has developed rapidly in recent years.The studies of the pre-erythrocytic vaccines,the blood-stage vaccines and the transmission blocking vaccines(TBVs)have made great sense currently in the world.The TBVs are designed to block malaria between human and mosquito hosts.It plays an important role in protecting population from disease,which has more far-reaching significance.However,only a few candidate antigens of TBVs have clear transmission blocking activity so far,and they have not achieved complete blocking effect.The complexity of the malaria parasite’s life cycle and the mechanism of the malaria parasite evades the immune response lead to large variations in its surface antigens.Immunization effect of monovalent vaccine is not ideal.Therefore,the development of multi-antigen malaria vaccine is the development trend currently.In our study,we screened Pbg37,PSOP25,and PSOP26,which were the candidate antigens of transmission blocking advantage obtained by the previous research group.Pbg37 is mainly expressed on the surface of gametophytes,and it has been shown to affect the development and maturation of male gametophytes by analyzing gene function.PSOP25 and PSOP26 are mainly expressed on the surface of ookinete.Gene function analysis shows that they significantly affect the formation of ookinete,as well as the number of oocysts and mosquito infection rate.We hypothesized that mixed or fusion vaccines would produce higher transmission blocking activity than single antigen vaccines.Therefore,we designed and expressed two fusion proteins consisting of the fragments of Pbg37 and PSOP25 and the fragments of PSOP25 and PSOP26.We expected that after immunizing mice with mixed or fusion protein antigens,the antibodies produced in the mice against the mixed or fusion antigens could react with the individual antigens,which proved that there was no immune interference between the two antigens.Both antibodies against mixed antigens and fusion antigens could recognize the native protein of P.berghei,and the transmission blocking activity of multivalent antibodies had a certain increase.This research provides new guidance and theoretical basis for the development of transmission blocking vaccines and multi-antigen vaccines.Methods:1)Plasmodium parasites,mice and mosquitoes.All animal experiments meet the requirements of the Animal Ethics Committee of China Medical University and have been approved by the committee.The strain of P.berghei ANKA was maintained from previous studies.Plasmodium parasites were injected into the blood system of mice through intravenous injection and then developed and proliferated after invading red blood cells.The female Balb/c mice,6-8 weeks old,were purchased from the Beijing Animal Institute.Anopheles stephensi(Hor strain)were reared in an environment with a temperature of 25°C and a humidity of 60-70%.2)Construction of protein expression vector.The expression regions were fragment of each full protein(Pbg37:26-88 aa,PSOP25:45-245 aa,PSOP26:50-254 aa).The total RNA extracting from Plasmodium,were reversed transcribed into c DNA as a template for gene amplification.After designing primers,Pbg37,PSOP25 and PSOP26 fragments encoding amino acids were obtained by PCR method,and then the overlap PCR was performed to amplify the Pbg37-PSOP25 and PSOP25-PSOP26 fusion fragments.The pET-32a(+)was used as the vector for protein expression.The PCR products and the vector were digested simultaneously by Bam H I and Not I and then were cloned to form a complete plasmid by ligase.3)Protein expression and purification.The plasmid with the correct sequence was transformed into Rosetta-gami B(DE3)strain.Expressions of protein were induced by 1 m M IPTG for 8 h at 20℃.After collecting the bacteria,the Ni-NTA agarose column were used for purifying the protein with His-Tag.The obtained protein was identified by 10%SDS-PAGE and Western Blot.4)Immunization program.There were nine Immunization groups including 1.WT,2.Trx-His,3.rPbg37,4.rPSOP25,5.rPSOP26,6.rPbg37+rPSOP25,7.rPSOP25+rPSOP26,8.rPbg37-PSOP25,9.rPSOP25-PSOP26.BALB/C mice(n=5)were injected with recombinant protein emulsified in the complete Freund’s adjuvant subcutaneously.Two subsequent booster immunization was carried out at two weeks interval.The serum was collected at 14 days after the final immunization,and the antibody titer was detected by Enzyme-linked immunosorbent assay(ELISA).5)Western Blot and Indirect Immunofluorescence Assay(IFA).Western Blot and IFA were used to detect the specific reaction between the polyclonal antibody against fusion protein and the native parasite protein.The antigens of schizont,gametocyte and ookinete extracting from the plasmodium were incubated with antiserum collecting from each immunization group first and then incubated with HRP-conjugated anti-mouse antibody.Protein bands were visualized by chemiluminescence using a Pierce ECL Western Blots Kit.For IFA,gametocyte,gamete and ookinete were incubated with immunization antiserum and then labeled with a fluorescent secondary antibody.A Nikon C2 fluorescence confocal laser scanning microscope were used to observe fluorescence.6)Evaluation of transmission blocking ability.The transmission blocking potential of each protein was tested by the blocking exflagellation assay and ookinete formation assay in vitro and direct mosquito feeding assay in vivo.For blocking exflagellation assay,gametocytes were cultured in the medium containing1:5/1:10 diluted antisera for 15 min and then the exflagellation centers were counted.The gametocytes as described above were then cultured for 24 h to quantified the level of ookinete formation in the medium.For mosquito feeding experiments,mice were immunized with each protein.On day 14 after the final immunization,mice were infected with 5×10~6parasites.Three days after infection,mice were fed on starved female A.stephensi.Oocyst density and mosquito infection rate were determined as described above.7)Statistical analysis.Statistical analysis was performed with the SPSS software,version 23.0.Antibody titer,exflagellation centers and ookinetes were analyzed by ANOVA analysis.The Oocyst intensity was analyzed by Mann-Whitney U test.The prevalence of infection was analyzed by Fisher’s exact test.P<0.05 was considered statistically significant.Results:1)Pbg37-PSOP25 and PSOP25-PSOP26 protein vector construction and protein expression.We screened three candidate antigens(Pbg37,PSOP25,PSOP26)to form two new dual-antigen vaccine(Pbg37-PSOP25,PSOP25-PSOP26).These candidates have great transmission blocking potential,which have been proved in previous studies.First,we constructed two protein expression vectors of fusion protein expression.Pbg37 is mainly expressed in the gametocyte and PSOP25 is expressed in the ookinete.These two genes were fused to construct a multi-stage fusion antigen vaccine.The other was to assemble PSOP25 and PSOP26,expressed at the same stage,to form a dual-antigen vaccine.We used a flexible linker to connected two antigen expression regions including Pbg37:26-88 aa(63 aa),PSOP25:45-245 aa(200aa)and PSOP26:50-254 aa(204 aa).After the two plasmids were transformed and expressed successfully,the results of SDS-PAGE gel and Western Blot showed that the protein size of rPbg37-PSOP25 and rPSOP25-PSOP26 with His-Tag were 51 kDa and66kDa,respectively.2)Immunization with recombinant proteins induces high antibody titer.Following prime and boost immunization,the mixed and fused antigens generated specific antibodies against individual antigens which the dual-antigen contained.As expected,immunization with individual antigens yielded only the antibodies specific for the immunization antigen.The OD value forPbg37 induced by the single antigen was similar with the mixed and fused antigens.The same results happened with PSOP25 and PSOP26.The results suggested that these domains do not exhibit antigenic competition.The specificity of immune sera was assessed using a western blot.Antibodies generated from mice immunized with Pbg37 in mixed or fused antigens recognize the rPbg37 at the band of 27 kDa.Antibodies against PSOP25recognize the rPSOP25 at 42 kDa and antibodies against PSOP26 recognize the rPSOP26 at 42 kDa.Antibodies against individual,mixed or chimeric proteins all recognized the rPbg37-PSOP25 at 51 kDa while recognized the rPSOP25-PSOP26 at66kDa.All results demonstrated that recombinant proteins induced strong immunogenic response in mice and produced specific antisera against the recombinant proteins 3)Recognition of the native protein in Plasmodium malaria.According to previous studies,we have confirmed that Pbg37 was localized in gametocytes,gametes,zygotes,and ookinetes but not in schizonts and PSOP25/PSOP26 was on the outer surface of the ookinetes.In our study,we determined whether sera from mice immunized with the mixed or chimeric proteins react to native proteins by western blotting.We observed that both the mixed and fused antibodies about Pbg37 and PSOP25 recognize a single band at～37 kDa of the gametocyte lysates while a dual band at the ookinete lysates,a deeper band at～40 kDa,a lighter band at～37 kDa,consisting with previous results.And other results showed that both the mixed and fused antibodies about PSOP25 and PSOP26 recognize two bands at～40 kDa and～90 kDa,consisting with previous results.To further characterize the specificity of the antibodies,an immunofluorescence assays was performed on gametocytes,gametes and ookinetes.We observed that antisera from the groups immunized with mixed or fused antigens including rPbg37+rPSOP25 and rPbg37-PSOP25 react to gametocytes,gametes and ookinetes,while antisera against rPSOP25+rPSOP26 and rPSOP25-PSOP26 react to gametes and ookinetes.4)Transmission-blocking activities of mixed and fused vaccines.To study the TB activities of the antisera,we first evaluated the effect of the antibodies both in vitro exflagellation centers and ookinetes formation inhibition assay.When wild type P.berghei gametocytes were incubated with the antisera at the 1:5 and1:10 dilutions in complete ookinete culture medium,male gametogenesis was counted under microscope after 15 min.Antisera against rPbg37,rPbg37+rPSOP25 and rPbg37-PSOP25 reduced the formation of exflagellation centers by 68%,68%and 70%at 1:5dilution and 63%,63%and 61%at 1:10 dilution compared to Trx-His group.Compared to control sera,antisera against rPSOP25 and rPSOP26 showed no differences in the formation of exflagellation centers.The result showed that the sera against mixed and fused antigens have a similar inhibition effect on the formation of exflagellation centers than anti-rPbg37 sera.The ookinete numbers were counted 24h after incubation,antisera against rPbg37,rPSOP25,rPSOP26,rPbg37+rPSOP25,rPbg37-PSOP25,rPSOP25+rPSOP26 and rPSOP25-PSOP26 reduced the formation of ookinetes by 68%,55%,49%,74%,77%,60%and 59%at 1:5 dilution and 60%,53%,42%,65%,67%,55%and 56%at 1:10 dilution compared to Trx-His group.The result showed that the sera against mixed and fused antigens have a stronger inhibition effect on the formation of ookinetes than individual sera.And the effect of immune sera on exflagellation and ookinete formation was concentration-dependent.To further evaluate TB activity of the antibodies in vivo,mice immunized with the respective recombinant antigens were infected with P.berghei and used for direct mosquitoes feeding.Compared to Trx-His control groups,the prevalence of infection and midgut oocyst density were significantly reduced.In this experiment,mosquitoes that fed on the mixed antigens rPbg37+rPSOP25 and rPSOP25+rPSOP26 achieving TRA of 75%and 66%(P<0.01),and infection prevalence of 80%and 82%,achieving TBA of 16%and 14%(P<0.01).The mean intensity were oocysts per midgut in rPbg37-PSOP25 immunized mice,achieving TRA of 77%(P<0.01),and the infection prevalence were 76%,achieving TBA of 20%(P<0.01).The mean intensity were oocysts per midgut in rPSOP25-PSOP26 immunized mice,achieving TRA of 66%(P<0.001),and the infection prevalence were 81%,achieving TBA of 15%(P<0.01).Conclusion:1)The molecular weights of the fusion proteins rPbg37-PSOP25 and rPSOP25-PSOP26 are 51kDa and 66kDa(contains the His-Tag).2)The antibodies obtained from the immune mixture and the fusion protein have higher antibody titers,and these domains do not exhibit antigenic competition.3)The antiserum obtained from the immune mixture and the fusion protein can react with the native protein of Plasmodium at a specific stage,and it produces an immune response against either individual antigen.4)The transmission blocking ability of rPbg37+rPSOP25 and rPbg37-PSOP25 to block the formation of oocyst was significantly higher than rPbg37and PSOP25.The transmission blocking ability of rPSOP25+rPSOP26 and rPSOP25-PSOP26 to block the formation of oocyst was significantly higher than rPSOP26.5)The transmission blocking activity of dual-stage vaccine was stronger than that of the multi-locus vaccine fused by the same stage protein.