The Screening Of Plasmodium Vivax-specific Antigens

Malaria is an anopheles mosquito-borne potentially fatal parasitic infectious disease. It is prevalent worldwide, including China, with the major specie present being Plasmodium vivax. Although the China Malaria Elimination Action Plan (MEAP) was introduced last year, the outbreak of P. vivax in central part of China during first decade of this century has meant that the malaria control status of China is fragile. The parasitemia and density of P. vivax are both low, but any missed diagnosis of malaria case may result in spreading by local transmission. Fast and accurate diagnosis of malaria is not only of benefit to treatment of patient, but also crucial for malaria control. The classic microscopy method requires special equipment, and is laborious and time consuming. More importantly, the accuracy and sensitivity largely depends on the experience of the individual technician. Using this method, the rate of missed diagnosis of malaria is high, and thus the high target set by MEAP is not achieved.In recent years, lateral flow-based Rapid Diagnostic Tests (RDTs) for malaria have been growing fast, and are becoming well established. RDTs have both high sensitivity and specificity, and can be performed without expensive equipment. The evaluation of test results is easy and the training time is reduced, thus RDTs are advantageous in field settings. The usefulness has been validated in falciparum malaria prevalent area. WHO give high regards to RDTs, as it already has held 4 rounds of evaluation programs and modified the guideline for use of RDTs, including recommending that all suspected patients with all age should be tested by parasitic diagnosis prior to antimalarial drug treatment. But until now, there are no RDTs suitable for vivax malaria prevalence areas, the major reason is lack of P. vivax antigens both of high sensitivity and specificity as the detection target. Given that P. vivax has such prevalence in China, improvement of the research on P. vivax-specific antigens for development new vivax malaria diagnosis RDTs should be consider as a high priority. The technique of in vitro continuous culture of P. vivax is not mature enough, and there is currently no way for providing abundant material for antigen research. And blood from vivax malaria infected patient is often contaminated with same amount or above host WBCs, which are difficult to remove. The experiments needed to be performed cannot be done because of a lack of the available purified parasites. These difficulties brought by vivax malaria-infected blood samples for research is one of the bottlenecks for breakthrough.Aldolase (ALD) is a key enzyme of the glycolytic pathway, and has shown desirable results as RDTs target in P. falciparum diagnosis. The sequence of P. vivax aldolase (PvALD) gene is available in GenBank. Clone and expression of PvALD to raise monoclonal antibody is a feasible approach for developing P. vivax-specific malaria RDT. Pan-specie specific malaria monoclonal antibody M26-32 can recognize 5 species of plasmodium including P. vivax, many ELISA experiments shown excellent results in detection of P. vivax. Base on these research results, to modify IgM class M26-32 into IgM subunit, making it more suitable for immunogold labeling is a worthwhile effort shortcut to realize new malaria RDT. In addition, designing primers based on genes of M26-32 targeted antigens is helpful to discover potential antigen genes by screening of P. vivax cDNA library.The formal publication of P. vivax genome provides the research community huge resources of information, and has inspired a new pathway to screening P. vivax specific antigens. The repetitive in protein sequence is one of the characteristics of plasmodium spp. The first has been largely used as a malarial diagnostic antigen, Plasmodium falciparum Histidine-Rich Protein-2 (PfHRP-2), is of extensive tandem repeats. Screening and data mining from the whole protein sequence of P. vivax genome is a novel method to find new P. vivax-specific antigens. In this research, based on the characteristics of malaria prevalence in China, andtake consideration of the needs of China malaria control for new generation diagnostic tools, multiple approaches were used to investigate the key scientific question for screening of P. vivax-specific antigen. This is done through bioinformatics methods to perform data mining of repetitive peptides information in P. vivax whole genome wide protein sequence database. The most Repetitive peptides with linear B epitope prediction were screened to assess the antigen probability and to classify the P. vivax specificity of each candidate peptide. Learning the successful experience from pLDH-based P. falciparum RDT, PvALD was chosen as a target for cloning and expression. Recombinant PvALD were used as antigen to raise monoclonal antibody for P. vivax diagnostic purpose. And based on both knowledge of M26-32 and newly established P. vivax cDNA library, M26-32 is modified into IgM subunit and labeled with immunogold, and screened for potential genes of M26-32 targeted antigens in P. vivax cDNA library. In addition, through cooperation with technicians from enterprise, a new type of leukocyte removal filter was developed for treatment of malaria infected blood, to provide malaria antigen research purified materials.PartⅠRepetitive protein sequences and linear B epitope prediction-based screening of Plasmodium vivax specific peptidesObjective To establish a method for data mining of repetitive protein sequences from P. vivax whole genome protein sequence database, and combine with linear B epitope prediction to screen for potential P. vivax specific peptides and validating mouse immunoreactivity to the peptides.Methods Download FASTA format V6.0 P. vivax protein sequence data from PlasmoDB, strip redundant data and reformat for SQL database. Designing software for searching of repetitive sequence by using Delphi language, setup a small peptide as seed, and count each seed peptide in the whole database in a character by character manner, and record all searching results in a new SQL table. After all searching is complete, extract top 1,000 seeds peptide depending on the repeat times, and submit all peptides one by one to BcePred website, combined with using four parameters to predict potential linear B epitope, and compare each peptide to make sure of all candidate antigen peptides of P. vivax specificity. Then synthesize 6 candidate peptides and conjugate with KLH to immunize BALB/c mice. Use peptide-ELISA to determine the titer of serum of immunized mice and serum of native infected P. vivax patients.Results The whole peptide sequences of P. vivax from V6.0 PlamsoDB database numbered 5,432. After input into SQL database and calculation of the repetitive peptide using 16 aa seeds mode with software designed by this research, there was about 3 million rows of data collected. The top 1,000 repeat peptides were extracted from the results, and subjected to 4-parameter prediction of linear B epitope. Twenty two peptides with 3 positive in 4 parameters were taken into classification analysis, and 6 peptides with P. vivax-specificity ware considered as candidate, then synthesized and conjugated to KLH to immunize mice. After four schemes of immunization, the serum of the immunized mouse was determined by peptide-ELISA, all 6 candidate peptides can induce high-titer antibody responses in mice from 1:9,000 to 1:81,000. And 20 serums from P. vivax patients were tested, and results showed that positive rate of 6 candidate peptides ranging from 5% to 40%.Conclusions A method for screening repetitive peptides from genome wide protein sequences was established. Six of the repetitive peptides generated by this method were predicted as candidate P. vivax specific antigen peptides by combination using multiple parameter linear B epitope prediction, and all 6 peptides can induce high-titer antibody response in mice.PartⅡCloning, expression and monoclonal antibody preparation of Plasmodium vivax aldolaseObjective To clone the coding domain sequence of P. vivax aldolase (PvALD). Prokaryotic and eukaryotic expression of recombinant PvALD protein and the preparation of monoclonal antibody against PvALD.Methods Design primers with restriction site according to PvALD coding domain sequence from a reference stain P. vivax Salvador I. Extract PCR template DNA from P. vivax infected patient. PCR products were inserted into pET32c and pPIC9k vector, and transformed E. coli DH5αstain respectively, then recombinant plasmids were extracted. pET32c-PvALD were transformed into E. coli BL21(DE3), then induced with IPTG, and the recombinant protein was purified by His-tag. pPIC9K-PvALD was Sac I-linearized and transformed into GS115, then induced with methanol, and the recombinant protein was purified. Prokaryotic and eukaryotic expression recombinant PvALD proteins were cross-used to immunize BALB/c mouse, and the strategy included swapping of intraperitoneal and subcutaneously routes. And the spleen cells of immunized mice were used to fuse with SP2/0 cells, and then PvALD antibody secreted hybridoma was screened.Results The PvALD PCR product is about 1.1 kb, and after restriction enzyme digestion, ligation, transformation, and blue-white selection, the two type recombinant plasmids were successfully constructed and verified by double digestions and sequencing. Prokaryotic plasmid pET32c-PvALD was transformed into BL21(DE3), and induced with IPTG, purified by His tag. The recombinant PvALD was about 60 kD. Eukaryotic plasmid pPIC9K-PvALD was Sac I-linearized and transformed into GS115, then induced with methanol, and the recombinant PvALD purified was about 40 kD. And both recombinant PvALD were used for mouse immunization and screening of hybridoma supernatants, with a total of 11 strains of hybridoma secreting monoclonal antibody against recombinant PvALD obtained. The ELISA results show that titers of culture supernatants varied from 1:320 to 1:1,280.Conclusions The P. vivax gene was cloned, and two type recombinant PvALD proteins were expressed and purified using prokaryotic and eukaryotic expression respectively. A total of 11 strains of hybridoma secreting monoclonal antibody against recombinant PvALD were obtainedPartⅢThe immunology characteristics of pan species malaria monoclonal antibody M26-32Objective To modify the IgM monoclonal antibody M26-32 into an IgM subunit. Use immunogold labeling of the native and modified M26-32 for use in malaria detection. Using P. falciparum M26-32 target antigen gene primers for PCR screening homologous gene in P. vivax cDNA library.Methods After thawing and cultivation, inoculate M26-32 hybridoma in BALB/c mice intraperitoneally and collect ascitic fluid. Purify M26-32 using Hitrap IgM Hp column, and lyse the IgM antibody into IgM subunit. After verification of titers by IFA, two types of M26-32 antibodies were labeled by immunogold, and tested with both P. falciparum and P. vivax soluble antigens. Primers were designed based on information of P. falciparum M26-32 target antigen gene fragments, the erythrocytic stage P. vivax cDNA library was screened by PCR, and amplified products were identified by sequencing.Results A total of 20 mice were used and about 80 ml ascitic fluid was collected. A highly purified M26-32 antibody was obtained by using HiTrap IgM Purification HP column. Native PAGE shows that L-cysteine can lyse the pentamer IgM into IgM subunit. IFA result shows no titer difference between two types of M26-32 monoclonal antibody. After labeling with immunogold, both two types M26-32 can recognize 1:100 diluted P. falciparum antigens and 1:10 diluted P. vivax antigens. Nine primer pairs were designed based on P. falciparum M26-32 target antigen gene fragments, and the P. vivax cDNA library was PCR screened. Amplification products were gel purified, and sequencing results show that 4 of 5 sequences were from vector of cDNA library, and 1 sequence contained P. vivax fragments. But after checking with 3 framed protein sequences, no known M26-32 target epitope sequences were found.Conclusions Established a method for modifying native IgM M26-32 monoclonal antibody into IgM subunit by using L-cysteine, and established a method for labeling both type M26-32 with immunogold, provided useful information for designing malaria RDT based on M26-32 monoclonal antibody. There is still need for further work in discovering potential M26-32 target antigen gene in P. vivax. Part IV The development and evaluation of leukocyte removal filter for purifying malaria-infected bloodObjective To develop a leukocyte removal filter for purifying malaria-infected blood. To evaluate the usage of new filters in malaria research.Methods Designing the plastic shells of the filter. The PBT (Polybutylene Terephthalate) melt-blown non-woven fabric modified with polar groups was used as the filter media. After above 99% leukocyte removal rate was achieved in laboratory. The new non-woven filter then was evaluated in P. vivax malaria prevalence area. Both WBCs removal rates and RBCs recovery rates was measured for comparing filter filtration and CF11 cellulose method by using same amount of P. vivax-infected blood samples. After treatment of both methods, samples were subjected to in vitro short term culture, and microscopy was used to observe the developing status of parasites. Plasmodium berghei-infected mouse blood and P. falciparum-infected human blood were filter filtrated as P. vivax samples, the leukocyte removal results were observed by microscopy.Results The leukocyte removal rate of filter with 3 layers non-woven fabric membranes for treatment of 10 ml health donor blood is 99.31%, which was achieving the design requirement, and was chosen for evaluation in field. Then it was chosen for evaluation in field. A total of 15 P. vivax-infected blood samples were treated with filter filtration method and CF11 method separately using the same sample (equal to 5 ml whole blood respectively). The WBC removal rate for filter method was 99.03%, which was better than CF11 method (98.41%, P<0.01). The RBC recovery rate for filter method was 95.48%, far better than CF11 method (87.05%, P<0.01). Fourteen in vitro short term culture result showed that after filter treatment, P. vivax parasite could develop the same as CF11 method, and no density, developing stage difference were fund between the two methods. Microscopy observation results show that before and after filter filtration, there was no obvious density, stage proportion and morphological changes were noticed in all P. vivax, P. falciparum and P. berghei samples, but the WBCs reduced heavily in thick film.Conclusions Successfully developed a new non-woven fabric leukocyte removal filter for purifying malaria-infected blood, suitable for treatment of P. vivax, P. falciparum-infected human blood and P. berghei-infected mouse blood. After field evaluation, filter method is better than CF11 method in both WBC removal and RBC recovery performance testing. Using filter method can provide finer P. vivax parasite material for molecular biology and antigen research. It is also suitable for pre-treatment of samples for P. vivax short term cultures. This filter is of both high performance in WBC removal and RBC recovery, and also faster and easier than CF11 method, thus better suited for field use.In summary, this research utilized a new bioinformatics approach, establishing a method based on searching repetitive sequences from P. vivax whole genome protein sequence database, combined this with linear B epitope prediction, in order to screen for P. vivax-specific peptides. By using molecular biology techniques, cloned, expressed and purified recombinant PvALD proteins from prokaryotic and eukaryotic system, and 11 stains of PvALD antibody secreting hybridoma were obtained. Utilizing immunological and molecular biological methods, pan-species malaria monoclonal antibody M26-32 were modified into IgM subunits, making it easier for labeling, and preliminarily screening P. vivax cDNA library in order to find a potential M26-32 target antigen gene. Building on materials science achievements, a new non-woven fabric leukocyte removal filter for purifying malaria-infected blood was developed, providing a high performance and easy-to-use tool for preparation finer P. vivax parasite material for molecular biology and antigen researches.