Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding Taenia solium oncosphere TSOL18 gene.

Cysticercosis due to larval cysts of Taenia solium, is a serious public health problem affecting humans in numerous regions worldwide. The oncospheral stage-specific TSOL18 antigen is a promising candidate for an anti-cysticercosis vaccine. It has been reported that the immunogenicity of the DNA vaccine may be enhanced through codon optimization of candidate genes. The aim of the present study was to further increase the efficacy of the cysticercosis DNA vaccine; therefore, a codon optimized recombinant expression plasmid pVAX1/TSOL18 was developed in order to enhance expression and immunogenicity of TSOL18. The gene encoding TSOL18 of Taenia solium was optimized, and the resulting opt-TSOL18 gene was amplified and expressed. The results of the present study showed that the codon-optimized TSOL18 gene was successfully expressed in CHO-K1 cells, and immunized mice vaccinated with opt-TSOL18 recombinant expression plasmids demonstrated opt­TSOL18 expression in muscle fibers, as determined by immunohistochemistry. In addition, the codon-optimized TSOL18 gene produced a significantly greater effect compared with that of TSOL18 and active spleen cells were markedly stimulated in vaccinated mice. 3H-thymidine incorporation was significantly greater in the opt-TSOL18 group compared with that of the TSOL18, pVAX and blank control groups (P<0.01). In conclusion, the eukaryotic expression vector containing the codon-optimized TSOL18 gene was successfully constructed and was confirmed to be expressed in vivo and in vitro. The expression and immunogenicity of the codon-optimized TSOL18 gene were markedly greater compared with that of the un-optimized gene. Therefore, these results may provide the basis for an optimized TSOL18 gene vaccine against cysticercosis.

[Malaria epidemiologic characteristics in Wuhe County of Anhui Province from 2009 to 2011].

OBJECTIVE: To understand the malaria epidemiologic characteristics in Wuhe County, Anhui Province from 2009 to 2011, so as to provide the evidence for formulating effective malaria control and prevention interventions. METHODS: The data of malaria cases from the reporting system and malaria epidemiological investigations were collected and analyzed statistically in Wuhe County from 2009 to 2011. RESULTS: The total number of malaria cases was 349 in Wuhe County from 2009 to 2011, and the incidence showed a downtrend. The sex ratio of patients was 1.42: 1 (205 males vs. 144 females). The ranks of patient age groups were 11-20 years old at the first and 31-40 at the second, and the youngest was 1 year old, and the oldest was 100 years old. The seasonal factor was observed clearly, malaria cases were rare during the period of January to March, the case numbers increased in April and reached the peak during the period of June to September, and the cases from November to December accounted for only 2.01% of the total cases. Among the 349 patients, there were only 2 patients (0.57%) living in urban areas and the rest 347 patients (99.43%) all living in rural areas. The incidence of the urban population was 0.08/10 000, and it was significantly lower than that of the rural population (1.78/10,000, P < 0.05). The highest incidences occurred in hill townships, Zhuding Town and Xiaoxi Town, the annual incidences were 5.53/10,000 and 4.78/10,000, respectively. The cases in hill townships accounted for 36.10% of the total cases. The most patients in these areas lived in brick or cement buildings without mosquito-proof doors or windows. They preferred sleeping outside during summer, and were generally lack of the malaria prevention knowledge. CONCLUSION: In Wuhe County, the malaria incidence is decreasing year by year, and the epidemiologic factors are related to the living conditions, mosquito-proof facility using, sleeping habit, and malaria awareness of the residents.

[Role of CD8+ T cells in the tumor growth delay induced by Toxoplasma gondii excreted-secreted antigen in B16F10 mouse melanoma model].

OBJECTIVE: To observe the role of CD8+ T cells in the tumor growth delay induced by Toxoplasma gondii excreted-secreted antigens (TgESA) in B16FI0 mouse melanoma model in the early stage. METHODS: TgESA were prepared by incubating T. gondii tachyzoites for 12 h in vitro. 15 C57BL/6 mice were randomly assigned to group A, B, and C (5 mice per group). Each mouse in group B and C was subcutaneously injected in right flank with 2 x 10(5) B16F10 cells. Mice in group C were intraperitoneally injected with TgESA (100 microl per mouse) at 7d after B16F10 cells injection. Mice of group A were only injected with PBS. On the 13th day after melanoma cell injection, the mice were sacrificed and spleen was removed. The percentage of CD8+ T cells in the spleen was analyzed by flow cytometry. CD8+ T cells were isolated from spleen cells by using immunomagnetic beads. The activity of CD8+ T cells against B16F10 melanoma cells was determined by LDH release assay at different effect-to-target cell ratios (2.5:1, 5:1, and 10:1). Other 30 C57BL/6 mice were randomly divided into group E, F, and G. Each mice were injected with 2 x 10(5) B16F10 cells. At the same time, mice in group F and G were simultaneously injected via the tail vein with CD8+ T cells isolated from mice in group B and C. Tumor growth, mortality and survival time of mice were observed and recorded during 35-d observation period. RESULTS: The percentage of CD3+CD8+T cells in the spleen cells of group C [(15.74 +/- 0.28)%] was significantly higher than that of group B [(14.18 +/- 0.27)%] and A [(13.86 +/- 0.13)%] (P < 0.05). At different effect-to-target cell ratios, the activity of CD8+ T cells against B16F10 cells in group C was significantly higher than that of group B (P < 0.05). The average time of tumor formation in group G [(14.9 +/- 1.2) d] was longer than that in group F [(11.9 +/- 0.7) d] and E [(9.4 +/- 1.2) d] (P < 0.05). The tumor size in these groups increased, but there was no obvious difference in the tumor growth rate among the three groups. The tumor size of group G was significantly smaller than the other two groups (P < 0.05). In group E, F and G, mice began to die on the 26th day, the 29th day and the 30th day after tumor inoculation, and the number of survival mice was 3, 5 and 7, respectively, at the 35th day after injection. CONCLUSIONS: TgESA may up-regulate the quantity and function of CD8+ T cell in B16F10 melanoma mouse model, which plays a role of delaying tumor growth in early stage.

[Sequence homology analysis on lactate dehydrogenase (LDH) of Plasmodium vivax Anhui isolates].

DNA in dried blood spots of 39 vivax malaria patients (2009-2010) from Anhui (Bengbu urban district and counties of Wuhe, Huaiyuan, Mengcheng and Lixin) was extracted. The Plasmodium vivax LDH (PvLDH) gene was amplified, cloned and sequenced. The sequences were subjected to NCBI Blast program. The results showed that the targeted DNA fragment size was 951 bp without difference among the 39 samples (accession No. GU078391), and was more than 99% homologous to the PvLDH sequences in other strains from GenBank. There was only one different amino acid in the protein sequences between the isolates from Anhui and EJEU60134 or MIA061251 strains.

[Recombinant expression and immunologic identification of bradyzoite-specific antigen BSR4 of Toxoplasma gondii].

OBJECTIVE: To express the recombinant BSR4 protein of Toxoplasma gondii Prugniaud (PRU) strain and study its immunologic characteristics. METHODS: The recombinant plasmid pET28a(+)-BSR4 was transformed into E. coli BL21, followed by expression of BSR4 induced by IPTG and its purification. The immunoreactivity of the recombinant protein BSR4 was analyzed by Western blotting with the sera from mice infected by PRU strain of T. gondii or normal mice as the first antibody. The 3H-TdR incorporation assay was performed to determine the proliferation of splenocytes in mice infected by PRU strain stimulated by BSR4, and the stimulation index (SI) was calculated. ELISA was used to evaluate the immunoreactivity of BSR4 protein, in which 20 sera from each of acute (anti-T. gondii IgG(-)IgM+) and chronic (IgG+IgM(-)) toxoplasmosis patients, and healthy people were tested. RESULTS: The recombinant BSR4 was induced and expressed. After denaturation, renaturation and purification, the soluble protein (Mr 45 000) was obtained and detected by anti-T. gondii serum with Western blotting. BSR4 in 1, 5, and 25 microg/ml induced the proliferation of splenocytes in mice infected by PRU strain with higher SI (1.13, 0.88, and 1.17) than that of control (0.46, 0.24, and 0.49, respectively, P<0.01). ELISA showed that the recombinant BSR4 specifically reacted with the sera of toxoplasmosis patients (IgG+IgM(-), 20/20), not with that of the acute cases (IgG(-)IgM+, 0/20). CONCLUSION: The recombinant BSR4 shows specific immunogenicity and immunoreactivity.

[Effects of excreted/secreted antigens of Toxoplasma gondii on CD4+ CD25+ Foxp3+ T cells and NK cells of melanoma-bearing mice].

OBJECTIVE: To explore the effects of excreted/secreted antigens (ESA) of Toxoplasma gondii on CD4+CD25+ Foxp3+ T cells and NK cells of melanoma-bearing mice, as well as the tumor growth. METHODS: B16F10 (denoted B16) tumor cells were cultured in complete medium and maintained by serial passage in vitro. The 2x 10(5) B16 tumor cells were injected into the right flank of the mouse to establish the tumor-bearing mice model. Mice were randomly divided into four groups, namely PBS, B16F10, PES/ESA and B16F10/ESA groups after ESA injections. On days 2, 4 and 6 post-ESA injection, the spleens were removed. The percentage of CD4+CD25+ Foxp3+ T cells and NK cells in splenocytes were determined by flow cytometry; the suppression functions of CD4+CD25+ Tregs and the NK cell activity were detected by WST-8 and LDH methods, respectively. The tumor growth of each group was measured. RESULTS: On Days 4 and 6 post-ESA injection, the percentages of CD4+CD25+ Foxp3+ T cells in splenocytes of the B16F10/ESA-injected mice decreased being (1.65 +/- 0.18)% and (1.56 +/- 0.17)%, respectively, and compared with those in the B16-injected mice [(2.47 +/- 0.10)% and (2.82 +/- 0.12)%], there were significant differences (both P values < 0.05). The inhibition of CD4+CD25+ Tregs of the B16F10/ESA-injected mice decreased markedly on Day 4 (50.03%) and Day 6 (50%) compared with those in the control (75.03% and 78.14%) post-ESA injection, there were significant difference (both P values < 0.05). The percentages of NK cells in splenocytes on Day 6 post-ESA injection [(3.58 +/- 0.07)%] was significantly higher than that of control [(2.61 +/- 0.13)%]. The activities of NK cells from B16F10/ESA - injected mice against B16 cells at different effect - to - target cell ratios (5 : 1, 10 :1, 20 : 1), increased significantly being 26.51%, 35.25%, 60.19%, respectively, while compared with those in the control (16.81%, 24.63% and 45.62%), there were significant differences (all P values < 0.05). In addition, the volume of the B16 tumors [(6 208.34 +/- 443.64)]mm3 was significantly smaller than that of control [(9 027.46 +/- 1 362.01)] mm3(P < 0.05) when measured at Day 35 post-tumor innoculation. CONCLUSIONS: T. gondii ESA can downregulate CD4+CD25+Tregs while upregulating NK cells of B16 tumor-bearing mice quantitatively and functionally, therefore plays a role in suppression of tumor growth.