Protective immunity induced by a DNA vaccine cocktail expressing TgSAG1, TgROP2, and the genetic adjuvant HBsAg against Toxoplasma gondii infection.

Toxoplasma gondii is an intracellular obligate parasitic protozoon that can infect all warm-blooded animals, causing zoonotic toxoplasmosis. So far, there is no commercial toxoplasmosis vaccine for human use. In the present study, we constructed a DNA vaccine cocktail which includes the surface protein (SAG1) and the rhoptry protein ROP2 denoted as pEGFP-N1-SAG1-ROP2. In order to improve the efficacy, HBsAg was used as a genetic adjuvant to construct pEGFP-N1-HBsAg-SAG1-ROP2. Two eukaryotic plasmids were transiently transfected into HEK293T cells and the expression was examined using fluorescence microscopy and western blotting. We then immunized Kunming mice intramuscularly with the DNA vaccine. After three immunizations, the immune response was evaluated by measuring antibody levels, cytokine production, percentages of CD4+ and CD8+ T lymphocytes, and the survival times of the T. gondii RH strain challenged mice. The results showed that the two DNA vaccines stimulated Th1 responses, and had a higher antibody titer, IL-2, IL-12, and IFN-γ levels, and percentage of CD4+ and CD8+ T lymphocytes than the control group. In addition, mice immunized with the pEGFP-N1-HBsAg-SAG1-ROP2 vaccine showed increased survival times compared with pEGFP-N1-SAG1-ROP2.

Characteristics of Imported Malaria and Species of Plasmodium Involved in Shandong Province, China (2012-2014).

Malaria remains a serious public health problem in Shandong Province, China; therefore, it is important to explore the characteristics of the current malaria prevalence situation in the province. In this study, data of malaria cases reported in Shandong during 2012-2014 were analyzed, and Plasmodium species were confirmed by smear microscopy and nested-PCR. A total of 374 malaria cases were reported, 80.8% of which were reported from 6 prefectures. Of all cases, P. falciparum was dominant (81.3%), followed by P. vivax (11.8%); P. ovale and P. malariae together accounted for 6.4% of cases. Notably, for the first time since 2012, no indigenous case had been reported in Shandong Province, a situation that continued through 2014. Total 95.2% of cases were imported from Africa. The ratio of male/female was 92.5:1, and 96.8% of cases occurred in people 20-54 years of age. Farmers or laborers represented 77.5% of cases. No significant trends of monthly pattern were found in the reported cases. All patients were in good condition after treatment, except for 3 who died. These results indicate that imported malaria has increased significantly since 2012 in Shandong Province, especially for P. falciparum, and there is an emergence of species diversity.

[Prokaryotic Expression, Purification and Immunological Characterization of Micronemal Protein 16 of Toxoplama gondii].

Objective: To prokaryotically express three gene fragments of micronemal protein 16 （TgMIC16） of Toxoplasma gondii, and analyze the immunoreactivity of the three recombinant protein products. Methods: Primers were designed for three fragments of TgMIC16 gene which encode proteins within the functional domain. Reverse-transcription PCR was used to generate cDNA from RNA, and the three fragments were amplified on the cDNA by PCR using the designed primers. The PCR products were double-digested, inserted into the pET-32a（+） plasmid, and transformed into Escherichia coli TOP10 cells. Plasmids extracted from positive clones were confirmed by BamHⅠ/HindⅢ double digestion and sequencing, and further transformed into E. coli Rosetta cells. Protein expression was induced by IPTG, and confirmed by SDS-PAGE. The expressed recombinant proteins were purified with Ni-NTA affinity chromatography and their immunoreactivity analyzed with Western blotting. Results: The amplified three fragments were 1 806, 1 290 and 855 bp in size. Double digestion and sequencing results confirmed the successful construction of the three recombinant plasmids. SDS-PAGE analysis showed successful expression of the three recombinant proteins (M(r) 88 000, 68 000 and 52 000, respectively）, in the form of inclusion bodies. Western blotting showed that the three purified recombinant proteins reacted with His monoclonal antibody and rabbit anti-T. gondii antibody. Conclusion: The three fragments within the functional domain of TgMIC16 are successfully expressed in prokaryotic expression system and show immunoreactivity.

[Detection of Genetic Mutations Associated with Drug Resistance in Imported Plasmodium falciparum].

Objective: To investigate the mutation of genes associated with drug resistance (Pfcrt, Pfmdr1, Pfdhfr and K13） in imported Plasmodium falciparum in Shandong Province. Methods: Blood was collected from 94 falciparum malaria cases who returned from Africa in 2014. Genomic DNA for P. falciparum was extracted from the blood samples and nested PCR was performed using primers specifically designed for Pfcrt, Pfmdr1, Pfdhfr and K13. The PCR products were sequenced. Gene mutations were analyzed by sequence alignment. Results: The 94 imported cases were from 18 African countries. Nested PCR was successful on DNA from all the blood samples except for Pfcrt amplification in one sample. Sequence analysis revealed three types of mutations Pfcrt K76T （36.6%, 34/93）, Pfmdr1 N86Y （21.3%, 20/94）, and Pfdhfr S108N （98.9%, 93/94） （χ2=127.5， P<0.05）. K13 C580Y mutation was not found. Co-occurrence of K76T, N86Y, and S108N was found in 6 blood samples （6.5%）, which were imported from Liberia（2）, Angola（1）, Equatorial guinea（1）, Congo（1）, and Guinea（1）. Co-occurrence of K76T and S108N mutations was found in 28 samples（30.1%）, and that of N86Y and S108N in 14 samples （15.1%）. Forty-four samples（47.3%） harbored S108N mutation only, and one sample was null for any of the mutations. Conclusion: There are mutations in Pfcrt, Pfmdr1, and Pfdhfr in imported Plasmodium falciparum in Shandong Province. No mutation was found for the K13 gene.

[Cloning and Bioinformatics Analysis of Toxoplasma gondii ROP21 Gene].

The full-length gene sequence of Toxoplasma gondii ROP21 (TgROP21) gene was amplified with PCR. The signaling peptide and transmembrane domain of TgROP21 protein were predicted by SignaIP and TMHMM online predictive sites, and the hydrophilicity and antigenic index of this protein were ananlyzed with DNAStar software. Meanwhile, the functional domains and tertiary structure were modeled by combined use of ExPASY and PRODATA online sites. As expected, the PCR results revealed one band at 2,022 bp. The signaling peptide, transmembrane domain, hydrophilicity, antigen index, functional domain and 3D structure of TgROP21 were successfully predicted. This work may provide a theoretical foundation for further verification of TgROP21 function.

[Extraction Method of Malaria Parasite DNA from Preserved Positive Blood Smears].

OBJECTIVE: To develop a method for DNA extraction from malaria parasites on preserved blood smears, to provide basis for research on malaria genetic traceability. METHODS: The improved DNA extraction kit (QIAamp DNA Mini Kit) was used to extract plasmodium DNA from 41 giemsa-stained blood smears, and the extraction was compared with that using the Chelex-100 and Na(2)HPO(4) methods. Nested PCR was used to amplify small subunit ribosomal RNA to identify Plasmodium parasite. The PCR products underwent sequencing and sequence alignment, to analyze the difference in PCR positive rates between blood smears prepared in the 1980s and in recent 10 years, between blood smears with and without deoil/decoloration, and between blood smears with different qualities. RESULTS: The total PCR positive rate for the improved kit method was 70.7% (29/41). The PCR positive rate for blood smears prepared in the 1980s and in recent 10 years was 78.6% (11/14) and 66.7% (18/27) respectively, with no significant difference (W=0.63, P>0.05). The PCR positive rate for blood smears with and with- out deoil/decoloration was 62.5% (15/24) and 82.4% (14/17) respectively, also with no significant difference (χ(2)= 1.89, P>0.05). However, the PCR positive rate was significantly higher in blood smears with high quality [93.3% (28/30)] than those with low quality [9.1%(1/1l)](=27.59, P<0.01). Sequence alignment showed that the PCR products were consistent with the target DNA fragments. However, DNA extracted using the Chelex-100 and Na(2)HPO(4) methods showed negative PCR results. CONCLUSIONS: DNA extracted from blood smears prepared in the 1980s using the improved Kit (QIAamp DNA Mini Kit) shows a high PCR positive rate. Besides, blood smear staining and use of oil for microscopic examination do not affect DNA extraction.

[Application of Nested PCR in the Diagnosis of Imported Plasmodium Ovale Infection].

OBJECTIVE: To identity Plasmodium ovale infection by 18S rRNA gene nested PCR. METHODS: Whole blood and filter paper blood samples of malaria patients in Shandong Province were collected during 2012-2013. The parasites were observed under a microscope with Giemsa staining. The genome DNA of blood samples were extracted as PCR templates. Genus- and species-specific primers were designed according to the Plasmodium 18S rRNA gene sequences. Plasmodium ovale-positive specimens were identified by nested PCR as well as verified by sequencing. RESULTS: There were 7 imported cases of P. ovale infection in the province during 2012-2013. Nested PCR results showed that the P. ovale specific band (800 bp) was amplified in all the 7 specimens. Blast results indicated that the PCR products were consistent with the Plasmodium ovale reference sequence in GenBank. CONCLUSION: Seven imported cases of ovale malaria in Shandong Province in 2012-2013 are confirmed by nested PCR.

[Research progress on polymorphism in the merozoite surface protein-3alpha gene of Plasmodium vivax].

The merozoite surface protein-3alpha of Plasmodium vivax (PvMSP-3alpha) is a surface protein that is expressed during the asexual blood stages. With a high polymorphism, PvMSP-3alpha gene has been used as a suitable epidemiology and genotype marker. Moreover, it might be an important malaria vaccine candidate. This paper summarizes the research progress on the molecular structure, gene polymorphism and genotypes of PvMSP-3alpha.

XPD codon 312 and 751 polymorphisms, and AFB1 exposure, and hepatocellular carcinoma risk.

BACKGROUND: Genetic polymorphisms in DNA repair genes may influence individual variation in DNA repair capacity, which may be associated with risk of hepatocellular carcinoma (HCC) related to the exposure of aflatoxin B1 (AFB1). In this study, we have focused on the polymorphisms of xeroderma pigmentosum complementation group D (XPD) codon 312 and 751 (namely Asp312Asn and Lys751Gln), involved in nucleotide excision repair. METHODS: We conducted a case-control study including 618 HCC cases and 712 controls to evaluate the associations between these two polymorphisms and HCC risk for Guangxi population by means of TaqMan-PCR and PCR-RFLP analysis. RESULTS: We found that individuals featuring the XPD genotypes with codon 751 Gln alleles (namely XPD-LG or XPD-GG) were related to an elevated risk of HCC compared to those with the homozygote of XPD codon 751 Lys alleles [namely XPD-LL, adjusted odds ratios (ORs) were 1.75 and 2.47; 95% confidence interval (CIs) were 1.30-2.37 and 1.62-3.76, respectively]. A gender-specific role was evident that showed an higher risk for women (adjusted OR was 8.58 for XPD-GG) than for men (adjusted OR = 2.90 for XPD-GG). Interestingly, the interactive effects of this polymorphism and AFB1-exposure information showed the codon 751 Gln alleles increase the risk of HCC for individuals facing longer exposure years (Pinteraction = 0.011, OR = 0.85). For example, long-exposure-years (> 48 years) individuals who carried XDP-GG had an adjusted OR of 470.25, whereas long-exposure-years people with XDP-LL were at lower risk (adjusted OR = 149.12). However, we did not find that XPD codon 312 polymorphism was significantly associated with HCC risk. CONCLUSION: These findings suggest that XPD Lys751Gln polymorphism is an important modulator of AFB1 related-HCC development in Guangxi population.

Protective immune response against Toxoplasma gondii elicited by a novel yeast-based vaccine with microneme protein 16.

Toxoplasma gondii is an obligate intracellular protozoan that can invade all eukaryotic cells and infect all warm-blood animals, causing the important zoonosis toxoplasmosis. Invasion of host cells is the key step necessary for T. gondii to complete its life cycle and microneme proteins play an important role in attachment and invasion of host cells. Microneme protein 16 (TgMIC16) is a new protective protein in T. gondii and belongs to transmembrane microneme proteins (TM-MIC). The TM-MICs are released onto the parasite's surface as complexes capable of interacting with host cell receptors. In the present study, we expressed the TgMIC16 protein on the surface of Saccharomyce cerevisiae (pCTCON2-TgMIC16/EBY100) and evaluated it as a potential vaccine for BALB/c mice against challenge infection with the RH strain of T. gondii. We immunized BALB/c mice both orally and intraperitoneally. After three immunizations, the immune response was evaluated by measuring antibody levels, lymphocyte proliferative responses, percentages of CD4+ and CD8+ T lymphocytes, cytokine production, and the survival times of challenged mice. The results showed that the pCTCON2-TgMIC16/EBY100 vaccine stimulated humoral and cellular immune responses. In addition, mice immunized with the pCTCON2-TgMIC16/EBY100 vaccine showed increased survival times compared with non-immunized controls. In summary, TgMIC16 displayed on the cell surface of S. cerevisiae could be used as potential vaccine against toxoplasmosis.

Expression of codon-optimized TgMIC16 in three Escherichia coli strains.

In a previous study, we found that rabbit anti-Toxoplasma gondii serum was capable of recognizing truncated T. gondii microneme protein 16 (TgMIC16), indicating that TgMIC16 is an essential antigenic T. gondii protein. However, the broad application of this recombinant protein is limited by its low expression level. In this study, we performed codon optimization of TgMIC16 by changing the codon-adaptation index from 0.22 to 1.0 without altering the amino acid sequence and expressed the optimized gene in three different Escherichia coli strains, followed by comparison of soluble recombinant-protein expression and yield. Our results showed that the recombinant protein rTgMIC16 was expressed as inclusion bodies in all three strains following optimization of induction parameters, and western blot analysis revealed the presence of a ~72-kD recombinant protein as a specific band following purification. A shuffle-expression strain was selected to amplify incubation products and induce expression, resulting in an overall rTgMIC16 yield of ~20 mg/L. These findings provide a basis for further investigation of TgMIC16 to elucidate its functions and interaction partners.

[Cloning and identification of ROP2 gene of Toxoplasma gondii].

OBJECTIVE: To amplify ROP2 from the genomic DNA of Toxoplasma gondii RH strain and construct eukaryotic expression plasmid pc-DNA3-ROP2. METHODS: Tachyzoites of T. gondii RH strain were collected and depurated to obtain genome. A pair of primers was designed and synthesized according ROP2 gene sequence. The gene fragment encoding ROP2 was amplified from the genomic DNA of T. gondii RH strain by means of PCR. It was then reclaimed and purified, and inserted into cloning vector pUCm-T. The recon was cut by EcoR I, Hind III, and the inserted ROP2 gene fragment was subcloned into pc-DNA3 eukaryotic expression vector using T4DNA ligase, followed by further PCR identification, double digestion via restrictive enzymes, and sequencing. RESULTS: The amplified specific gene fragment of ROP2 was about 1.7 kb in length. The gene fragment cloned and subcloned into pc-DNA3 was correct, and the eukaryotic expression plasmid contained ROP2 gene fragment was successfully constructed. CONCLUSION: The recombinant plasmid pc-DNA3-ROP2 was successfully constructed.

[Studies on the immuno-protection of ROP2 nuclei acid vaccine in Toxoplasma gondii infection].

OBJECTIVE: To study the protective effect of ROP2 nuclei acid vaccine in mice. METHODS: Forty-two BALB/c mice were divided into three groups. Each mouse in experiment group was injected with 50 microg recombinant plasmid pc-DNA3-ROP2 through musculus quadriceps fexoris. In control groups, each mouse was injected with 50 microg blank plasmid pc-DNA3 and with 50 microl PBS respectively. All mice were immunized for three times with an interval of three weeks. The volume was doubled for the final injection in the two plasmid groups. Blood, spleens and lymph nodes of 4 mice in each group were taken for the detection of CD4+, CD8+ T cells and cytokines 2 weeks after the final immunization. The rest mice in 3 groups were challenged with 500 tachyzoites of Toxoplasm gondii RH strain for further observation. RESULTS: The vaccine induced strong cellular and humoral immune response. The titer of antibody in serum was high after inoculation and recognized ROP2 protein antigen expressed in vitro. The lymphocyte phenotype was analyzed. CD4+ T cells proliferated sharply (69.5+/-3.4)%, and the ratio of CD4/CD8+ increased considerably by (4.69+/-1.32)% (P<0.01). The level of IL-2, IL4, IL-6, IL-12, IFN-gamma and TNF in serum and cultured supernatant of spleen cells and lymph cells was higher in the experiment group than that in control groups, especially in serum. 88.9% mice in the experiment group were protected 180 hours after the challenge of T. gondii. The death time of mice in experiment group was delayed and the survival time was prolonged in comparison to that in control groups with a significant difference (P< 0.01). CONCLUSION: The recombinant ROP2 nuclei acid vaccine shows fair immunogeni-city and obviously produces immuno-protection.

[Cloning, expression, purification and identification of Toxoplasma gondii SAG2 gene in Escherichia coli].

OBJECTIVE: To construct a recombinant plasmid containing surface antigen 2(SAG2) gene of Toxoplasma gondii and express it in Escherichia coli. METHODS: The truncated SAG2 gene was amplified from the genomic DNA of T. gondii RH strain and cloned into plasmid pGEX-4T. Then the recombinant pGEX-4T-SAG2 was induced by IPTG and expressed in E. richia col BL21. The expressed proteins were analyzed by SDS-PAGE and purified, and the immunogenicity of the product was analyzed by Western blotting. RESULTS: The amplified SAG2 gene was about 561 bp, which was accorded to the expectation. The recombinant plasmid was constructed successfully by digested with double restriction enzyme and confirmed with DNA sequencing. SDS-PAGE and Western blotting showed the molecular weight of SAG2 fusion protein was about 47 ku, and the protein could be identified by GST-tag antibody. CONCLUSION: The truncated SAG2 gene of T. gondii has been successfully cloned and expressed in E. coli BL21 cells, and the recombinant protein has immunogenicity.

Establishment of lymphatic filarial specific IgG4 indirect ELISA detection method.

OBJECTIVE: To establish the lymphatic filarial specific IgG4 indirect ELISA detection method and develop the kits. METHODS: ELISA and the developed specific IgG4 reagent was used to explore the best way for detecting filarial specific IgG4. Combined with the production process of commercialized enzyme immunoassay kit to develop economical lymphatic filarial specific IgG4 test kit, and to explore the value of the kit in the laboratory. RESULTS: We determined the most optimal detective antigen was Malay adult filarial antigen and the optimal concentration of coating antigen was 1.0 µg/ml. The appropriate serum dilution was 1:20 to 40 and the work titers of specific IgG4 agents was 1:800. We determined the optimal reaction time for substrates and developed a reproducible and stable detection kit with sensitive and specificity, which was easy to operate. CONCLUSION: We successfully established the lymphatic filarial specific IgG4 indirect ELISA detection method and developed the kits with good reproducibility and stable result, which should be widely applied.

Construction and identification of Complex DNA vaccine of hepatitis B and Toxoplasma gondii.

OBJECTIVE: To construct and identify multi-gene recombinant expression vector pcDNA3-HBsAg-p30-ROP2. METHOD: Primers were designed according to the gene sequences of restriction enzyme cutting site of recombinant pcDNA3-p30-ROP2 and hepatitis B surface antigen (HBsAg). The target fragment of HBsAg was amplified and cloned to expression vector pcDNA3-p30-ROP2 by restriction enzyme digestion and ligation. The recombinant expression vector pcDNA3-HBsAg-p30-ROP2 was identified by PCR detection, followed by enzyme restriction and sequencing. RESULTS: The target fragment of HBsAg was successfully amplified, and the multi-gene eukaryotic expression vector pcDNA3-HBsAg-p30-ROP2 was established. PCR detection and restriction enzyme digestion showed that the length of the target fragment was consistent with the theoretical value. The recombinant expression vector contained the complete sequences of p30-ROP2 compound gene and HBsAg. CONCLUSION: Multi-gene recombinant expression vector pcDNA3-HBsAg-p30-ROP2 was successfully established. The constructed expression vector could be used to develop multi-gene nucleic acid vaccines.

[Survey of intestinal parasitic infections and related knowledge and behavior of residents in Jiaodong area of Shandong Province].

OBJECTIVE: To understand the status of intestinal parasitic infections and the related knowledge and behavior in residents of Jiaodong area of Shandong Province, so as to provide the evidence for making an appropriate preventive and control strategy. METHODS: A total of 18 villages from 6 counties in Jiaodong area were selected as investigation sites according to the stratified sampling method. The feces samples of the permanent residents aged above 3 years were collected and examined by Kato-Katz technique to find the intestinal parasite eggs, and the children under 12 years old were examined by the method of cellophane anal swab to detect the Enterobius vennrmicularis eggs. In addition, 50 households in each survey sites were randomly selected to investigate the basic family situation and the condition of awareness on prevention knowledge and formation of correct behavior of residents by using a structured questionnaire. RESULTS: Totally 6 163 residents involved in the feces examinations, and the total infection rate of intestinal parasites was 6.91%. The infection rates of Trichuris trichiura, Ascaris lumbricoides and hookworm were 6.56%, 0.62% and 0.21%, respectively. The infection rate of E. vermicularis in children under 12 years old was 0.51%. The eggs of Clonorchis sinensis and Taenia solium were not found in this survey. The awareness rate of knowledge about preventing parasitic diseases was 49.54%. The formation rates of washing hands before eating, washing hands after using the toilet, never eating raw fruit and vegetable without washing clean, never working in the field with bare feet, and never drinking unboiled water were 97.78%, 91.95%, 88.81%, 92.42% and 86.48% respectively. CONCLUSIONS: The infection rate of intestinal parasites is low in Jiaodong area, but there is a significant difference among different counties. The awareness rate of knowledge about preventing parasitic diseases is low, but the formation rate of healthy behavior is high. In the future, the health education and the strategy of taking medicine among the key population should be enhanced, and the project of reconstructing safe water supply and lavatory should be advanced.

[Epidemiological analysis of malaria prevalence in Shandong Province in 2012].

OBJECTIVE: To understand the characteristics of malaria prevalence and epidemic in Shandong Province in 2012 so as to provide the evidence for improving the work of the elimination of malaria. METHODS: The epidemiological data of malaria cases collected from the Disease Surveillance Information Reporting System of Chinese Center for Disease Control and Prevention were analyzed with the descriptive epidemiological method for epidemiological characteristics of malaria. RESULTS: A total of 93 malaria cases were reported in Shandong Province in 2012 with the incidence of 0.097 per 100 thousand, with a reduction of 19.83% as compared to 2011. There were 93 imported cases which decreased by 4.12% compared with 97 cases in 2011 and it was the first year that there was no local infection. Jining, Qingdao and Weihai cities reported more cases, with 62.37% (58/93) of the total number of the whole province. Totally 93.55% of malaria cases were imported from Africa, most from Equatorial Guinea, Nigeria and Angola. There were 3 cases of imported ovale malaria firstly reported. CONCLUSIONS: There were no local malaria cases reported in Shandong Province in 2012, but the imported malaria prevention and control was still not optimistic. In order to achieve the goal of malaria elimination in Shandong Province, it needs to continue to strengthen epidemic management, professional training and work supervision, strengthen management, advocacy and detection on the floating population, and explore multisectoral coordination mechanisms.