Identification,Expression And Evaluation Of Two Plasmodium Vivax Sexual Stage Antigens As Transmission-Blocking Vaccine Candidates

Objective: Malaria is the most common parasitic disease and a widespread infectious disease in tropical regions.Malaria control meathods,such as insecticide-treated mosquito nets,spraying of insecticides,and artemisinin combination therapy,have collectively contributed to a significant reduction in malaria morbidity and mortality.Despite recent successes in reducing the global burden of malaria,the disease is still a major global health problem.According to the WHO World Malaria Report in 2019,there were 228 million malaria patients worldwide in 2018,and the total number of deaths dropped to 405,000.But with the global epidemic of COVID-19,some antimalarial-fighting efforts were interrupted.The WHO World Malaria Report in 2021 pointed out that in 2020 malaria was endemic in 85 countries,with global malaria infections rising to 241 million and deaths reaching 627,000.Now,critical 2020 milestones of WHO’s global malaria strategy have been missed,and without immediate and dramatic action,the 2030 targets of control and eliminate malaria will not realise.In addition to the impact of COVID-19,the emergence of drug-resistant parasites and drug-resistant mosquitoes poses a challenge to the control and elimination of malaria.The prevention and treatment of malaria is still one of the scientific problems that have received elevated attention.The Malaria Eradication Research Agenda（mal ERA）emphasizes hat the key to controlling malaria transmission is the development of new control methods to effectively block malaria transmission,and vaccines are undoubtedly the best weapon to accomplish this task.Malaria vaccines are classified into pre-blood stage vaccines,blood stage vaccines and transmission-blocking vaccines（TBVs）based on the life cycle of malaria parasites.Pre-blood stage vaccines and blood stage vaccines can reduce the infection rate and clinical morbidity of malaria,but they are limited by the pressure of human immunity and the relatively wide range of gene polymorphisms,which casuing disadvantages.TBVs were designed to target the sexual developmental of palsmodium,the antibodies of sexual stage antigens can block the subsequent development of parasites in the mosquito midgut,thus halting the transmission of malaria parasites.It is an important research direction for elimination of malaria.However,despite considerable efforts,no effective TBVs have been successfully developed and only a few candidate antigens have entered the preclinical stage.Research of malaria TBVs is in urgent need.Among the five Plasmodium species that cause human malaria infection,Plasmodium falciparum（P.falciparum）and Plasmodium vivax（P.vivax）are the most common types.Although less virulent than P.falciparum,P.vivax still poses a significant burden in Asia and the Americas.Some malaria patients are asymptomatic but still infectious to mosquitoes and possibly be an important reservoir that contributes to the sustained transmission of malaria.Because of the difficulty in culturing P.vivax in vitro,coupled with experimental conditions and ethical restrictions,the development of P.vivax TBVs is relatively slow.Almost all the P.vivax TBVs in clinical studies were derived from homologs of P.falciparum vaccine candidate antigens.There is an urgent need to develop novel TBVs candidate antigens.An important task in the development of novel P.vivax TBVs is to search for candidate antigens that can block Plasmodium transmission via mosquitos.A good vaccine candidate antigen should have the following characteristics:（1）they should be located on the cell surface to increase their access to immune system surveillance,（2）they must be antigenic,（3）they must have one or more transmembrane regions to facilitate expression,（4）they should be conserved sequences among different species of Plasmodium,and（5）they must be nonallergenic.In addition,essential antigens that may play an important role in the life cycle of P.vivax are considered as good targets for vaccine development.In recent years,with the identification of Plasmodium genome and the progress of multiple transcriptomics and proteomics studies,reverse vaccinology has become an important strategy for screening malaria vaccine candidate antigens.The basic idea of reverse vaccinology refers to the screening of candidate antigens in the whole parasite genome,using the Plasmodium genome as a platform to clone,express and purify recombinant proteins.It should be stage-specific,extramembrane expressed antigens that meet the structural requirements of vaccine proteins and affect the life cycle of parasites.Purified antigens were evaluated in vivo and in vitro,and the protective antigens were selected for vaccine research.Reverse vaccinology,which is more efficient and targeted,is a promising direction malaria vaccine research.Due to the importance of P.vivax TBVs and the urgent need for the development of novel vaccine candidate antigens,our group use the strategy of reverse vaccinology to screen TBVs candidate antigens from the whole Plasmodium genome.First,the Plasmo DB database are used to identify genes that are transcribed with high levels in the sexual stage（gametophytes and ookinetes）with transmembrane region and signal peptide.It may affect the transcription of functional transcription factors.Then the protein structure,subcellular localization and epitopes of the candidate antigens will be predicted to determine whether the target protein matched the structural characteristics of the vaccine antigen.Predict the post-transcriptional translation and folding characteristics of candidate antigens to in-depth analysis the structure and function of candidates.It can provide the basis for the expression of recombinant protein.Finally,use the blood samples from P.vivax infected patients to test the transmission blocking ability of potential antigens and their gene polymorphisms.Based on these method,our study will provide a new theoretical and experimental basis for the fight against P.vivax through searching novel TBVs candidate antigens.Methods:1.Bioinformatic analysis.The database Plasmo DB was searched according to the basic characteristics of TBV.The search strategy was that candidites need to have highlevel transcription in the gametophyte phase/ ookinetes phase;a signal peptide;and a transmembrane region.Based on the transcriptomic data of the functional transcription factors in the sexual stage in the database,the restriction conditions were further set to determine whether the target gene might be a functional gene that affects the transcription of important ranscrip factors.The function of selected genes were predicted.BLAST was used to compare gene sequences.Clustal W was used to analyze the homology of conserved between species.MAGA5 was used for phylogenetic tree analysis.Amino acid sequence were downloaded from Plasmo DB.The first,second and tertiary structure of the protein was predicted.Kolaskar and Tongaonkar were used for epitope analysis.Tm Hmm2.0 was used to analyze the transmembrane region.Through protein phosphorylation,disulfide bond and glycosylation prediction databases,the phosphorylation,glycosylation site and disulfide bond status of the target protein were predicted.The Anaphylactic analysis were predicted based on their amino acid sequences.2.Expression and purification of recombinant protein.The target gene fragments of recombinant protein were obtained by base synthesis after codon optimization.The gene fragments were linked to the p PIC9 K plasmid,and the recombinant plasmid was identified by double enzyme digestion and sequencing.The recombinant plasmid was electrotransfected into Pichia pastoris GS115.Transfection-positive bacteria were identified by colony PCR.The positive strains were cultured with yeast culture medium,induced by methanol,and centrifuged in a Backman centrifuge bottle,and the supernatant was filtered out.Finally,the recombinant protein was purified with a Ni-NTA column.The eluted and dialyzed samples were detected by SDS–PAGE,and the target protein bands were detected.A glutathione S-transferase（GST）protein was expressed as a negative control.3.Animal immunization and collected of antisera.To generate antisera against the two recombinant proteins,we injected female BALB/c mice（n = 10）subcutaneously with purified recombinant protein or GST control protein emulsified in complete Freund’s adjuvant.The mice were then given two more booster immunizations with the same recombinant proteins emulsified in incomplete Freund’s adjuvant at 2-week intervals.Finally,10 days after the third immunization,the antisera in each group of mice were collected via cardiac puncture and pooled.4.Analysis the immunogenicity of recombinant protein.The collected mouse antiserum was used as the primary antibody to evaluate the immunogenicity of the recombinant protein by Western-Blot analysis.The antigenicity of recombinant protein was evaluated by Elisa to detect the antibody titer in serum of immunized mice.5.Collection of blood samples from P.vivax infected patients.The human experimental protocol was approved by the Ethics Committee of the School of Tropical Medicine,Mahidol University,Thailand.In the summer of 2018,samples were collected from patients in Tak Province,Thailand and subsequent transmission blocking tests were conducted.Eligible for inclusion in this study were patients with clinical symptoms of P.vivax malaria,blood smear positive for P.vivax,older than 18 years and nonpregnant patients.After the patient signed the informed consent form voluntarily,5-10 ml of venous blood was collected and heparinized before starting anti-malaria treatment.Thus blood samples from P.vivax infected patients were obtained.6.Purify red blood cells containing P.vivax gametophytes.The blood samples of P.vivax infected patients were mixed with 47% Nycodenz-supplemented RPMI 1640 medium and centrifuged at 500 g for 25 min.After centrifugation,the parasite-infected erythrocytes could be obtained at the gray interface in the middle of the mixture.7.IFA detected the natural antigenicity of recombinant proteins.The purify red blood cells containing P.vivax gametophytes were used to make thin smears,which were fixed and incubated with recombinant protein antiserum.The slides were then incubated with fluorescein isothiocyanate（FITC）-conjugated goat anti-mouse antibodies.Fluorescence images were observed with a fluorescence microscope.8.Quantification the transmission blocking activity of recombinant protein with DMFA.Antisera from mice immunized with recombinant protein or the GST control protein were diluted with heatinactivated AB+ serum obtained from healthy donors in Thailand（1:1,v/v）.Erythrocytes of P.vivax patients were mixed with diluted sera（1:1,v/v）and incubated at 37 °C for 15 min.Each reconstituted infected blood sample was then introduced to a glass feeder and kept at 37 °C.One hundred starved mosquitoes were allowed to feed on the blood mixture for 30 min at 37 °C through the membrane feeder.After several hours,fully engorged mosquitoes were identified,isolated,and kept on a 10% sucrose solution in cotton balls at 20 °C at 80% relative humidity for 1 week.Twenty mosquitoes from each group were randomly dissected on day 7 after blood feeding.Mosquito midguts were stained with 5% mercurochrome,and oocysts were counted.The infection prevalence,which is the proportion of oocyst-positive infected mosquitoes,was used to determine transmission blocking activity.The intensity of infection,i.e.the number of oocysts per mosquito midgut was used to determine transmission reducing activity.9.Collection of P.vivax DNA samples.100μL of fingertip blood was spotted on Whatman filter paper,dried and stored in a compressed plastic bag for subsequent identification of gene polymorphisms of candidate proteins.The P.vivax DNA was obtained with a DNA extraction kit.10.Genetic polymorphisms analysis recombinant protein.Using P.vivax DNA as template,DNA fragments encoding recombinant protein were amplified by PCR.The primers were designed based on P.vivax Sal-1.All amplified DNA products were purified and sequenced.11.Statistical analyses.Statistical analyses were performed using SPSS version 22.0 software.Student t-test was used for comparison of antibody titers between the control and immunization groups.The intensity of infection was compared using the Mann–Whitney U-test.The infection prevalence was compared using Fisher’s exact test.P values of < 0.05 were considered to be statistically signifcant.Results:1.Screening and bioinformatics analysis of candidate antigens.Two P.vivax TBVs candidate antigens were screened from the whole Plasmodium genome: PVX₀98655（PvPH）and PVX₁01120（Pv SOP26）and they were named PvPH and Pv SOP26.They were predicte to fit the basic characteristics of TBVs,which were transcripting at the sexual stage,including transmembrane region and signal peptide and conserved among different species of Plasmodium.Transcriptomics in the database showed that PvPH and Pv SOP26 were related to the functional transcription factors.They may be the essential antigens in the sexual stage of Plasmodium.By predicting protein structure,subcellular localization and antigen B cell epitopes,PvPH and Pv SOP26 were predicated to be consistent with the structural characteristics of vaccine candidate proteins.They all contained an N-terminal signal peptide and several low-complexity regions.After excluding the signal peptide and comprehensively referring to factors such as antigenic determination,22-304 aa of PvPH and 30-272 aa of Pv SOP26 were selected for expression.The predicted molecular weights of PvPH and Pv SOP26 were 32 k Da and 29 k Da,respectively.The prediction of post-translational modification and folding characteristics of recombinant proteins revealed that r PvPH and r PvPH contain multiple phosphorylation,glycosylation sites,all of which are predicted to be nonallergenic proteins.The recombinant fragment of PvPH was predicted to contain disulfide bonds.2.Expression and identification of recombinant candidate proteins.The recombinant plasmids pvph-p PICK and pvsop26-p PICK of eukaryotic expression vectors were successfully constructed and transferred into Pichia pastoris GS115.After induction by methanol,r PvPH and r Pv SOP26（³2 k Da and ²9 k Da）was detected with a high yield.A Ni-NTA column was used to purify the recombinant protein,SDS–PAGE detection was performed on the eluted and dialyzed samples,and the target protein band was clearly observed.3.Identification immunogenicity of recombinant proteins.After an initial immunization and two enhanced immunizations,the target recombinant proteinimmunized mouse antiserum was collected.Western blot analysis showed that the antisera to r PvPH and r Pv SOP26 correctly identified the recombinant proteins respectively.ELISA results showed that after the third immunization,compared with the anti-GST control mouse serum,the mouse anti-r PvPH and r Pv SOP26 serum-specific antibody levels were significantly increased,and the antibody titer exceeded 1:320,000.Blood samples of P.vivax infected patients were collected in endemic areas in Thailand,and the gametophytes infected red blood cells were successfully purified with 47% Nycodenz.IFA results showed r PvPH mice antisera showed strong fluorescence signal on the surface of female and male gametophytes of P.vivax.4.Quantification of TB activity of the antisera of recombinant proteins.Blood samples of 3 P.vivax-infected patients were collected in Thailand’s malaria endemic area.DMFA was carried out with P.vivax patients blood mixed with recombinant protein antisera using laboratory-reared An.dirus mosquitoes.The results showed that the antiserum of r Pv SOP26 significantly reduced the oocyst density in mosquitoes fed serum from two P.vivax malaria patients and slightly reduced the infection rate in these two groups of mosquitoes.The r PvPH antiserum had no significant effect on mosquito oocyst density or infection rate.5.Gene polymorphism analysis candidate proteins.The gene fragments of the candidate antigens were amplified from the DNA of Thailand P.vivax with PCR primers designed according to the gene sequence of P.vivax Sal-1.The amplified DNA sequence was aligned with the Sal-I sequence.No amino acid mutation was found in the three patients with r PvPH,and the same amino acid mutation was found in r Pv SOP26,K263 N,I355S and L403 I.Conclution:1.PvPH and PvSOP26 were predicated to be essential antigens in the sexual stage of P.vivax and fit protein structure characteristics of vaccine antigens.2.The recombinant proteins r PvPH and r Pv SOP26 expressed by the pichia pastoris expression system showed good immunoreactivity in mice and P.vivax.3.The polymorphisms of PvPH and Pv SOP26 were limited in the DNAs extracted from Thailand P.vivax.4.The r Pv SOP26 mouse antisera showed strong transmission-blocking function in samples of P.vivax patients from Thailand.Pv SOP26 could be a promising TBV candidate for P.vivax.