[The world's first malaria vaccine: Hope and challenge].

The RTS,S/AS01 is a subunit malaria vaccine against the pre-erythrocytic stage of Plasmodium falciparum. After over 30 years of research and development and clinical trials, this vaccine has been recommended by the WHO for use among children living in highly malaria endemic areas. Although the RTS, S/AS01 vaccine suffers from problems of a low protective efficacy (about 30%), need of four doses and short duration of protective immunity, this malaria vaccine is expected to save tens of thousands of children's lives, and avoid tens of millions of malaria cases annually, because there have been tens of thousands of childhood deaths due to malaria recently. The introduction of the RTS, S/AS01 vaccine is therefore, widely accepted as a milestone in the history of battle against malaria, which brings a hope to contain malaria and even eventually eliminate malaria. Although there are still multiple challenges in the development of a satisfactory malaria vaccine, the success of the RTS, S/AS01 malaria greatly facilitates the progress towards the development of parasitic disease vaccines, and a more perfect malaria vaccine deserves expectations.

[A novel isothermal amplification assay improves the capability for the field rapid detection of parasitic diseases].

With the rapid development of molecular biology, the isothermal amplification technique has been used for the nucleic acid detection of parasites and other pathogens due to its high efficiency and rapid and simple procedures, and has become an important tool to promote the field detection and control of parasitic diseases. Recombinase-aided isothermal amplification assay (RAA), a novel isothermal amplification technique, which is simple and easy to perform, rapid for field detection, no need for high-end equipment, and rapid field detection, may amplify the target gene fragments within 5 to 20 min under an isothermal condition (usually 37 to 42 ℃) and achieve a real-time observation of the amplification results. RAA has been successfully employed for the nucleic acid detection of a wide range of parasites and other pathogens to date, and has shown a high sensitivity and specificity. Notably, such an assay is suitable for the large-scale field detection in non-lab environments, and is therefore considered to have a potential value of application in rapid field detections.

[Specific antibodies against recombinant MSP1 of Plasmodium falciparum strongly inhibit the parasite growth in vitro].

In order to produce large amounts of protein for vaccine trials, a synthetic msp1-42 gene was inserted into Pichia pastoris expression vector and the plasmid was introduced into Pichia pastoris SMD1168 by electroporation. The expressed MSP1-42 was secreted into the protein-free medium. To measure the conformational properties of MSP1-42,16 monoclonal antibodies (11 recognizing conformational epitopes) were allowed to interact with the Pichia-derived MSP1-42, and all antibodies specific for conserved and K1 protype interacted with the protein. Interestingly, three monoclonal antibodies (e.g. 9.8, 13.1 and 7.3), that were shown not to interact with CHO-derived MSP1, could interact with the Pichia-derived MSP1-42. Rabbits were immunized with recombinant MSP1-42 formulated with CFA adjuvant four times. The rabbits were bled on the day 3 after last immunization, and total IgG isolated by protein A column from the immunized rabbits was shown to strongly inhibit the parasite growth in vitro dose-dependently, whereas IgG from rabbit with adjuvant had no inhibition.

Preliminary study of the distribution of the toxic elements As, Cd, and Hg in human hair and tissues by RNAA.

In order to study the relationships between trace element concentrations of hair and internal body burdens, a radiochemical NAA technique has been used for determination of the elements As, Cd, and Hg in autopsy samples of liver, kidney-cortex, lung, and hair from 24 male persons who died by accident. High significant positive correlations were observed between the As concentration in hair and in kidney-cortex, and between Cd and Zn concentrations in kidney-cortex. The contents of Cd, both for lung and kidney-cortex, were related to the smoking habits of the subjects.

[Inducible expression of MSP1 gene of Plasmodium falciparum by a tetracycline-controlled promoter].

OBJECTIVE: To express the entire MSP1 gene of Plasmodium falciparum and its C-terminal 42 kDa fragment using a tetracycline-controlled PLtetO-1 promoter. METHODS: The entire MSP1 gene and 42 kDa fragment gene were cloned into the plasmid of pZE11, and transformed into E. coli DH5 alpha Z1. Restriction enzyme analysis, SDS-PAGE and Western blotting were used to examine two recombinant plasmids and their expression in E. coli DH5 alpha Z1. RESULTS: The recombinant plasmids of pZE11/MSP1 and pZE11/MSP1-42 were constructed successfully. The expressive products about 190 kDa and 42 kDa of two genes in E. coli DH5 alpha Z1 were identified by SDS-PAGE and Western blotting. CONCLUSION: Tightly controlling expression of the MSP1 gene in E. coli is essential to reduce the toxicity of the product to its host cells as well as to provide a feasibility to construct Salmonella vaccine strain which can inducibly express the important malarial vaccine candidate gene.

[Induced expression of the variable region of AMA-1 from Plasmodium falciparum].

OBJECTIVE: To express the variable region of AMA-1 gene from Plasmodium falciparum in Escherichia coli. METHODS: Genomic DNA of FCC1/HN was used as template and the variable region of AMA-1 gene was amplified by polymerase chain reaction(PCR). The PCR products were digested by endonuclease BamH I and Hind III, cloned into pBlu2KSP. The nucleotide sequences of the variable region of AMA-1 gene were determined by sequencing. The AMA-1 gene fragment was subcloned into plasmid pQE, expressed in E. coli and induced by IPTG. The fusion product as identified by SDS-PAGE gel electrophoresis and Western blotting were proceeded with anti-AMA-1 sera from rabbit. RESULTS: The size of the variable region of AMA-1 gene from FCC1/HN was 506 bp and encoded 168 amino acids. On SDS-PAGE gel dyed with Coomassie brilliant blue R250, no specific protein band can be discerned, but Western blotting proceeded with anti-AMA-1 sera from rabbit demonstrated that the specific protein band was about 23.0 kDa. CONCLUSION: The variable region of AMA-1 gene from FCC1/HN was able to be expressed in E. coli and analysis of Western blotting demonstrated that the AMA-1 fussion protein contained specific antigenic epitopes.

Investigation of recombinant Schistosoma japonicum paramyosin fragments for immunogenicity and vaccine efficacy in mice.

Schistosoma japonicum paramyosin, a 97 kDa myofibrillar protein, is a recognized vaccine candidate against schistosomiasis. To improve its expression and to identify protective epitopic regions on paramyosin, the published Chinese Schistosoma japonicum paramyosin cDNA sequence was redesigned using Pichia codon usage and divided into four overlapping fragments (fragments 1, 2, 3, 4) of 747, 651, 669 and 678 bp, respectively. These gene fragments were synthesized and expressed in Pichia pastoris (fragments 2 and 3) or E. coli (fragments 1 and 4). The recombinant proteins were produced at high level and purified using a two-step process involving Ni-NTA affinity chromatography and gel filtration. BALB/c mice were immunized subcutaneously three times at 2-week-intervals with the purified proteins formulated in adjuvant Quil A. The protein fragments were highly immunogenic, inducing high, though variable, ELISA antibody titres, and each was shown to resemble native paramyosin in terms of its recognition by the anti-fragment antibodies in Western blotting. The immunized mice were subjected to cercarial challenge 2 weeks after the final injection and promising protective efficacy in terms of significant reductions in worm burdens, worm-pair numbers and liver eggs in the vaccinated mice resulted. There was no apparent correlation between the antibody titres generated and protective efficacy, as all fragments produced effective but similar levels of protection.

[Immunization of mice with plasmid DNA against malaria and regulation of antigen expression by tetracycline-controlled promoter].

Sequence of MSP1-31 of Plasmodium falciparum was constructed into eukaryotic expression vector pTRE, which could be repressed by tetracycline (Tc) and resulted in recombinant plasmid pTRE-31. The plasmid was injected into the quadriceps muscle of BALB/c mice with Tc responsive plasmid pTet-off to measure specific antibodies. The MSP1-31 prokaryotic expressed protein was used as antigen in ELISA. Results showed that mice orally administered by Tc had a seroconversion rate of 7.1% (1/14) 4 weeks after injection, whereas the control mice had a seroconversion rate of 100% and the titers of antibody were raised continusly within 12 weeks. The study suggested that the recombinant plasmids pTRE-31/pTet-off could efficiently induce humoral response against MSP1-31 of malaria. Moreover this immune response was controlled by Tc and was reversible after withdrawal of Tc dilivery. The induction of antibody by removing Tc at the fourth week after injection indicated that DNA vaccine could remain in mice and capable of expressing antigen for at least 4 weeks.

[Expression of chimeric protein of Plasmodium falciparum in yeast recombinant Pichia pastoris in cell-high-density fermentation].

The effects of cultural conditions, which were fermentation period when methanol was as sole carbon source, methanol concentration, range of pH, on the expression of the chimeric protein of Plasmodium falciparum in genetically engineered methylotrophic Pichia pastoris were investigated by shake flask experiments in this paper. The results showed: (1) fermentation period with methanol inducement is about 96 hours; (2) the optimum methanol concentration as sole carbon source is 10 g/L; (3) the range of pH is from 6.0 to 7.0. On the base of above experimental results the cell high-density fermentation had been done on the FMG-5L fermentor with multi-sensors. The results showed that the cell optical density (OD600) can reached 550 and the maximum expression level of target proteins was 780 mg/L which was 4 times higher or more than the shake flask culture's.