Studies Of Sequence Variation And Immunoprotective Efficacy Of Toxoplasma Gondii Rhoptry Protein 38

Since Toxoplasma gondii was discovered in 1908, we are known T. gondii and toxoplasmosis for more than a century. Toxoplamosis can be transmitted by mother to child transmission (MTCT), oral transmission and blood-borne transmission. Due to the broad range of its hosts (almost all warm-blooded animals) and the diversity of transmission routes, a third of the world’s population have been infected with toxoplasmosis, and the trends is increasing every year. Most immune-competent people show no symptoms after infection, but so far, no drug could cure toxoplasmosis. Clinlic treatment of toxoplamosis depends on the use of chemical drugs such as sulfonamides. While these drugs could control acute T. gondii infection with some adverse effects, they cannot eliminate chronic infection. Therefore, studies of vaccines against toxoplasmosis have become a focus of research. Development of T. gondii vaccines has a long history, starting from the whole parasite vaccines, to vaccines based on specific components of the parasite, and to genetically engineered vaccines and DNA vaccines. Candidate antigens of DNA vaccines are usually related to T. gondii virulence and invasion, such as rhoptry, microneme or dense granule secretions. T. gondii rhoptry protein 38 (TgROP38) regulates MAPK signal transduction pathway. Its expression varies significantly in different T. gondii genotypes, and is upregulated remarkedly during the transmission from tachyzoites to bradyzoites.In the present study, we examined sequence variation in TgROP38 gene among T. gondii isolates from different hosts and geographical regions. The complete ROP38 gene from 13 T. gondii isolates were amplified and sequenced. Sequence alignments showed that the lengths of the entire ROP38 gene ranged from 2646bp to 2650bp, and the sequence variation was 0.2-1.1% among the 13 T. gondii isolates. Phylogenetic analysis of ROP38 sequences using Bayesian inference (BI) showed that the clustering of the 13 T. gondii isolates were not consistent with their respective genotypes.We then predicted biological characterisatics of TgROP38 utilizing bioinformatics softwares. The TgROP38 ORF sequence was amplified by PCR from the genomic DNA of T. gondii RH strain. After sequencing, the nucleotide sequence of TgROP38 gene was translated into amino acid sequence and was analyzed by bioinformatics softwares. TgROP38 protein was predicted to contain multiple transmembrane and hydrophilic regions and several B cell epitopes.Thirdly, TgROP38 gene was inserted into eukaryotic vector pVAX I and formed the DNA vaccine pVAX-ROP38. After pVAX-ROP38 was transfected into HEK 293 cells, indirect immunofluorescence assay (EFA) was used to detect the expression of pVAX-ROP38 in vitro. Mice in the experimental group were intramuscularly injected by pVAX-ROP38 and those received pVAX I, PBS and nothing as controls. After three times injection at two week intervals, all groups were challenged with 1000 tachyzoites of the virulent T. gondii RH strain and 10 cysts of the PRU strain, respectively. The results showed mice inoculated with pVAX-ROP38 vaccine had a higher level of IgG antibodies and IFN-y and IL-2 cytokines, activated Thl-type immune responses, produced the high level of CD3+CD4"CD8+and CD3+CD4+CD8" T lymphoproliferative response. In the model of acute infection, the average survival time of mice in pVAX-ROP38 group (8.1d± 0.75) was no statistically different compared to that in PBS, pVAX I and blank control groups which died within 7d. However, in the model of chronic infection, the brain cyst reduction in the pVAX-ROP38 group reached 76.6%, which was significantly decreased in contrast to controls.Finally, we evaluated the immune protection of mixed rROP38 and rROP18 proteins which were encapsulated in 50:50 poly (lactide-co-glycolide) (PLG) slow-released microparticles. We constructed PLG-rROP38 and PLG-rROP18 microparticles. Mice in the experimental groups were injected with rROP38, PLG-rROP38, rROP18, PLG-rROP18, rROP38-rROP18 and PLG-rROP38-rROP18, respectively and those received PBS, PLG-PBS and blank group were taken as controls. After the last immunization, humoral and cellular immunity related indexes were examined as that for pVAX-ROP38 vaccine. Six weeks after the last immunization, all groups were challenged with 10 cysts of the PRU strain. The results showed that immunization of mice with protein vaccines could elicit a higher level of IgG antibodies and IFN-y and IL-2 cytokines, activated a mixed Thl-Th2 type immune responses, produced the high level of CD3+CD4-CD8+ and CD3+CD4+CD8-T lymphoproliferative response. In the model of chronic infection, the number of brain cysts decreased 81.26% in the PLG-rROP38-rROP18 group, compared with that of the controls.In conclusion, this study amplified TgROP38 gene successfully, and revealed a low level (0.2-1.1%) of sequence variation among different T. gondii isolates. TgROP38 has multiple transmembrane and hydrophilic regions and several B cell epitopes. Both DNA vaccine and protein vaccine based on TgROP38 induced significant immunoprotection against challenge infection with the low virulent PRU strain, indicating that TgROP38 is a potential good candidate antigen against chronic T. gondii infection. PLG extended the duration of slow-release of protein vaccines. These results provide good foundation for further development of vaccines against chronic T. gondii infection.