Protective immunity against Toxoplasma gondii induced by DNA immunization with the gene encoding a novel vaccine candidate: calcium-dependent protein kinase 3

BMC Infectious Diseases Protective immunity against Toxoplasma gondii induced by DNA immunization with the gene encoding a novel vaccine candidate: calcium- dependent protein kinase 3 Nian-Zhang Zhang 1 Si-Yang Huang 1 Dong-Hui Zhou 1 Jia Chen 1 Ying Xu 0 Wei-Peng Tian 2 Jing Lu 4 Xing-Quan Zhu 0 1 3 0 College of Animal Science and Technology, Anhui Agricultural University , Hefei, Anhui Province 230036 , PR China 1 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Lanzhou, Gansu Province 730046 , PR China 2 College of Veterinary Medicine, Northeast Agricultural University , Harbin, Heilongjiang Province 150030 , PR China 3 College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University , Daqing, Heilongjiang Province 163319 , PR China 4 College of Veterinary Medicine, South China Agricultural University , Guangzhou, Guangdong Province 510642 , PR China Background: Toxoplasma gondii can infect almost all warm-blood animals including human beings. The plant-like calcium-dependent protein kinases (CDPKs) harbored by T. gondii are involved in gliding motility, cell invasion, egress and some other developmental processes, and so have been implicated as important virulence factors. Methods: In the present study, we constructed a DNA vaccine expressing T. gondii CDPK3 (TgCDPK3) and evaluated its protective efficacy against T. gondii infection in Kunming mice. The gene sequence encoding TgCDPK3 was inserted into the eukaryotic expression vector pVAX I, and mice were immunized with pVAX-CDPK3 intramuscularly. Results: The results showed that mice immunized with pVAX-CDPK3 developed a high level of specific antibodies and a strong lymphoproliferative response. The significantly increased levels of IFN-, IL-2, IL-12 (p70) and IL-23 and high ratio of IgG2a to IgG1 antibody titers indicated that a Th1 type response was elicited after immunization with pVAX-CDPK3. Furthermore, the percentage of CD4+ T cells in mice vaccinated with pVAX-CDPK3 was significantly increased. After lethal challenge with the tachyzoites of the virulent T. gondii RH strain, the mice immunized with pVAX-CDPK3 prolonged the survival time from 10 days to 24 days (13.5 4.89) compared to untreated mice or those received PBS or pVAX I which died within 7 days (P < 0.05). In chronic infection model (10 cysts of the T. gondii PRU strain), the numbers of brain cysts of the mice immunized with pVAX-CDPK3 reduced significantly when compared with those in control groups (P < 0.05), and the rate of reduction could reach to about 50%. Conclusions: TgCDPK3 can generate protective immunity against acute and chronic T. gondii infection in Kunming mice and is a promising vaccine candidate for further development of an effective vaccine against T. gondii. Toxoplasma gondii; Toxoplasmosis; TgCDPK3; DNA vaccine; Protective immunity - Background Toxoplasma gondii, an obligate intracellular protozoan parasite, is responsible for toxoplasmosis in a wide range of hosts including humans, mammals, birds and marine mammals [1-5]. T. gondii infection in immune-competent individuals is rarely symptomatic, but toxoplasmosis occurred in fetus and immunocompromised hosts may result in severe disease or even lethal damage [5-7]. Meanwhile, the infection can cause serious economic losses to the livestock industry, especially in sheep and goats, as the course of abortion, stillbirth and neonatal loss [8], and also the infected animals are major sources of T. gondii transmission to humans [4,6]. No available chemical treatments could completely eliminate T. gondii in infected animals, so immunoprophylaxis is considered to be high priority for prevention and control of the parasite [9,10]. Although the only licensed vaccine based on the attenuated-live T. gondii S48 strain (Toxovax) can be used to prevent the incidence of abortion in sheep [11], it is limited to be further explored in food-producing animals or humans in view of the safety concerns on its reverting to a virulence wild type. The current efforts have been made on the development of DNA vaccines due to the superiority of much safer than live-type vaccines, as well as their ability to induce primarily Th1 cell-mediated immune and CD8+ cytotoxic T cells (CTL) responses [12,13]. A family of calcium-dependent protein kinases (CDPK) is known as key effectors in regulating calcium related signaling pathways in apicomplexan, which control a diverse array of functions in the life cycle including gliding motility, cell invasion, egress and some other developmental processes that occur at distinct stages in their complex life [14]. TgCDPK3, a characteristic member of CDPKs, is localized to the parasite periphery in intracellular and extracellular parasites and partially to the apical end of the intracellular parasite [15]. The TgCDPK3 knockout strain showed fewer parasites per vacuole than parental strains, which implied that the gene can partially influence the division of T. gondii [15]. However, no studies have evaluated the immunogenicity of TgCDPK3 and its potential as a vaccine candidate against T. gondii infection. In this context, the objectives of the present study were to examine the various immune responses in mice induced by DNA immunization with a eukaryotic plasmid expressing TgCDPK3, and to evaluate the potential of TgCDPK3 as a vaccine candidate against infection with two different genotypes of T. gondii in Kunming mice. Methods Animals Specific-pathogen-free (SPF) female inbred Kunming mice of 68 weeks old were purchased from Center of Laboratory 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 Peoples Republic of China. This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. LVRIAEC2012-011). Parasites T. gondii RH and PRU strains were used in this study. Tachyzoites of the highly virulent T. gondii RH strain (Type I) were propagated by serial intraperitoneal passage in Kunming mice. The peritoneal fluid of mice was centrifuged for 10 min at 1 000 g at 4C to remove the cellular debris and then re-suspended in sterile phosphatebuffered saline (PBS). The obtained tachyzoites were also used for Toxoplasma lysate antigen (TLA) preparation according to our previous studies [16] and total RNA extraction was followed by the instruction of the RNAprep Pure Tissue Kit (TIANGEN, China) manual. Cysts of the PRU strain (Type II) were maintained in the laboratory by oral passage of infective brain homogenate in Kunming mice. Construction of the eukaryotic expression plasmid The complete open reading frame (ORF) of TgCDPK3 was amplified by revers (...truncated)