Evaluation of immune responses in mice after DNA immunization with putative Toxoplasma gondii calcium-dependent protein kinase 5.

Evaluation of Immune Responses in Mice after DNA Immunization with Putative Toxoplasma gondii Calcium-Dependent Protein Kinase 5 Nian-Zhang Zhang,a Si-Yang Huang,a Ying Xu,a,b Jia Chen,a Jin-Lei Wang,a Wei-Peng Tian,c Xing-Quan Zhua,d State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, People’s Republic of Chinaa; College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui Province, People’s Republic of Chinab; College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang Province, Chinac; College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province, People’s Republic of Chinad Toxoplasma gondii can cause serious public health problems and economic losses worldwide. Calcium-dependent protein kinases (CDPKs) are key mediators of T. gondii signaling pathways and are implicated as important virulence factors. In the present study, we cloned a novel T. gondii CDPK gene, named TgCDPK5, and constructed the eukaryotic expression vector pVAXCDPK5. Then, we evaluated the immune protection induced by pVAX-CDPK5 in Kunming mice. After injection of pVAXCDPK5 intramuscularly, immune responses, determined with lymphoproliferative assays and cytokine and antibody measurements, were monitored, and mouse survival times and brain cyst formation were evaluated following challenges with the T. gondii RH strain (genotype I) and the PRU strain (genotype II). pVAX-CDPK5 effectively induced immune responses with increased specific antibodies, a predominance of IgG2a production, and a strong lymphocyte proliferative response. The levels of gamma interferon (IFN-␥), interleukin 2 (IL-2), and IL-12(p70) and the percentages of CD3ⴙ CD4ⴙ and CD3ⴙ CD8ⴙ cells in mice vaccinated with pVAX-CDPK5 were significantly increased. However, IL-4 and IL-10 were not produced in the vaccinated mice. These results demonstrate that pVAX-CDPK5 can elicit strong humoral and cellular Th1 immune responses. The survival time of immunized mice challenged with the T. gondii RH strain (8.67 ⴞ 4.34 days) was slightly, but not significantly, longer than that in the control groups within 7 days (P > 0.05). The numbers of brain cysts in the mice in the pVAX-CDPK5 group were reduced by ⬃40% compared with those in the control groups (P < 0.05), which provides a foundation for the further development of effective subunit vaccines against T. gondii. I nfection with the obligate intracellular protozoan parasite Toxoplasma gondii has worldwide importance (1, 2, 3). The parasite can infect human beings and virtually all food production and companion animals and thus is able to cause serious public health problems (2, 4, 5, 6). T. gondii infection in fetuses and in immunodeficient individuals, including AIDS patients, can result in severe disease and even death (1, 7, 8). When the primary T. gondii infection occurs during pregnancy, it can lead to miscarriage, severe neonatal malformations, and ocular complications in the fetus (1, 9). T. gondii infection in animals not only is a veterinary problem causing abortion in sheep and goats but also represents a real risk for humans via ingestion of tissue cysts and oocysts (8, 10, 11). Currently, no available drug treatments can eliminate T. gondii cysts from infected hosts. Furthermore, the chemotherapeutic agents can cause consumer concerns about chemical residues in meat and also the emergence of drug-resistant parasites following long-term use (12). So, immunoprophylaxis would be extremely valuable for prevention of human and animal toxoplasmosis (13, 14). However, after more than 20 years of effort, only one licensed vaccine (Toxovax) can be used to prevent abortion in sheep, and it is based on the live attenuated tachyzoites of strain S48 (14, 15). In recent years, DNA vaccination has been demonstrated to elicit a predominantly Th1 immune response: inducing CD4⫹ T-lymphocyte and CD8⫹ cytotoxic T-lymphocyte (CTL) responses against the administered antigen (14, 16) and several T. gondii DNA vaccine candidates evaluated using this administration route (17, 18, 19, 20, 21, 22) have shown effective protection against T. gondii infection. Also, DNA vaccines are promising tools in the development of safe and effective vaccines against T. 924 cvi.asm.org Clinical and Vaccine Immunology gondii infection in both humans and animals (14), and thus it would be valuable to identify novel T. gondii antigens for use in DNA vaccination. As signaling mediators of calcium-related signaling pathways, calcium-dependent protein kinases (CDPK) can control a diverse array of functions in the life cycle of apicomplexans, including gliding motility, cell invasion and egress, and some other critical biological processes (23). Our unpublished data and previous studies showed that both TgCDPK1 and TgCDPK3 (22) are promising vaccine candidates that can elicit protective immunity against acute and chronic T. gondii infection, but the immunogenicity of other CDPK members is not yet known. In the present study, we cloned a novel putative CDPK gene, named TgCDPK5, from the T. gondii RH strain and then constructed the eukaryotic expression vector pVAX-CDPK5. The aims of the present study were to evaluate the various immune responses induced by pVAX-CDPK5 in Kunming mice and to analyze the potential of TgCDPK5 as a vaccine candidate against Received 18 February 2014 Returned for modification 17 March 2014 Accepted 22 April 2014 Published ahead of print 30 April 2014 Editor: P. P. Wilkins Address correspondence to Si-Yang Huang, , or Xing-Quan Zhu, . Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/CVI.00059-14 p. 924 –929 July 2014 Volume 21 Number 7 Immune Responses in Mice Induced by T. gondii CDPK5 infection with the virulent RH strain of T. gondii in Kunming mice. MATERIALS AND METHODS Mice and parasites. Six- to 8-week-old specific-pathogen-free (SPF) female Kunming mice were purchased from the Center of Experimental Animals, Lanzhou Institute of Biological Products, Lanzhou, China. All mice were handled in strict accordance with the good animal practice requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China. The virulent T. gondii RH strain and the brain cyst-forming PRU strain were used in this study. Tachyzoites of the T. gondii RH strain (type I) were propagated by serial intraperitoneal passage in Kunming mice. If needed, the peritoneal fluid of mice was centrifuged for 10 min at 1,000 ⫻ g at 4°C and then resuspended in sterile phosphate-buffered saline (PBS). The obtained tachyzoites were used for total RNA extraction following the instructions in the RNAprep pure tissue kit (Tiangen, China) manual and also to prepare Toxoplasma lysate antigen (TLA) as described in our previous study (24). Cysts of (...truncated)