Toxoplasma gondii vaccines and uses thereof

#Toxoplasma gondii is an obligate intracellular parasite capable of infecting most warm-blooded vertebrates and many nucleated cell types. Parasite transmission occurs orally through ingestion of tissue cysts or sporozoites from feline feces in contaminated soil, food, and water. Infection typically results in an asymptomatic primary infection that leads to a chronic latent infection affecting 30% of the world's population (Carruthers (2002) Acta Trop. 81:111-122). Following oral ingestion of tissue or oocyst cysts, parasites are released into the gut mucosa where they infect host cells and transform into the rapidly replicating tachyzoite stage. Rapidly replicating tachyzoites disseminate widely throughout the host reaching most organs and the brain. Host immune pressure is thought to trigger differentiation of tachyzoites into slow growing bradyzoites and development of tissue cysts. Despite the potent Th-1 acquired immunity that is elicited by primary infection, tissue cysts persist in immune privileged sites such as the brain for the life of the host. The reactivation of bradyzoites to tachyzoite differentiation in brain cysts leads to recrudescent and life threatening Toxoplasmic encephalitis in AIDS patients (Luft and Remington (1992) Clin. Infect. Dis. 15:211-222). T. gondii primary infections in pregnancy also lead to spontaneous abortion or severe CNS damage in neonates. As T. gondii is the 3rd leading cause of food-born illness in the U.S., it is a significant human pathogen and therefore understanding the mechanisms underlying the development of protective immunity in response to infection is of high importance to development of vaccines.

#T. gondii is now a widely recognized model for host response mechanisms. During active infection, T. gondii induces a potent systemic Th-1 inflammatory response that results in life long CD8+ T cell-mediated immune control of the infection. Infection triggers the innate response through a MyD88-dependent pathway resulting in IL-12-independent production of IFN-γ by NK and T cells leading to the recruitment of neutrophils and macrophages to the site of infection (Scanga, et al. (2002) J. Immunol. 168:5997-6001; Mun, et al. (2003) Int. Immunol. 15:1081-1087; Scharton-Kersten, et al. (1996) Exp. Parasitol. 84:102-114). Concomitant with the innate response, the development of the acquired Th-1 response is driven by secretion of IL-12 from neutrophils, macrophages and DCs that increases inflammatory cell infiltration, activates APCs and enhances production of IFN-γ by T cells and NK cells leading to the cell-mediated immune control (Bennouna, et al. (2003) J. Immunol. 171:6052-6058; Gazzinelli, et al. (1994) J. Immunol. 153:2533-2543).

#Certain mechanisms of the immune response and key mediators of host immune control have been defined. Previous studies of host responses have typically used replicating and infectious strains of T. gondii that widely disseminate and cause extensive host tissue destruction and associated host-derived inflammatory responses. Other immune response models are based on studies using whole parasite antigen or parasite components (Scanga, et al. (2002) supra; Mun, et al. (2003) supra; Scharton-Kersten, et al. (1996) supra; Bennouna, et al. (2003) supra; Gazzinelli, et al. (1994) supra; Aliberti, et al. (2000) Nat. Immunol. 1:83-87). These host response and vaccine models reveal that immunization with weakened, but living and invasive T. gondii parasites results in complete protection against lethal challenge infections (Waldeland and Frenkel (1983) J. Parasitol. 69:60-65; Snzuki and Remington (1988) J. Immunol. 140:3943-3946; Bourguin, et al. (1998) Infect. Immun. 66:4867-4874; Kasper, et al. (1985) J. Immunol. 134:3426-3431).

#FIG. 1 shows the percent survival of mice immunized with cps1-1 knock-out via different routes of administration. FIG. 1A, C57Bl/6 mice were unimmunized or immunized with either 1×s.c., 2×s.c., 1× i.p. or 2× i.p. and challenged 1 month after final immunization with 103 RH and percent survival was measured. FIGS. 1B and 1C, C57BL/6 mice were unimmunized or immunized once with either cps1-1 knock-out alone or DC, pMAC, or PEC loaded ex vivo for 12 hours with cps1-1 knock-out. Two months (FIG. 1B) or 6 months (FIG. 1C) post-i.v. immunization, mice were challenged with 102 tachyzoites of RH i.p. and percent survival was measured out to 30 days post-challenge. Data represents one experiment performed with 6 mice per immunization group. In FIGS. 1B and 1C, statistical significance was calculated using Kaplan-Meier product limit test.

#FIG. 2 shows the effect of antibody depletion of T cells, lack of B cells, and adoptive transfer of immune cells on survival against lethal challenge. C57Bl/6 wild-type and μMT mice were immunized following an established protocol. One month after final immunization, wild-type mice were treated with either control Ig or antibody specific for CD4, CD8, or both CD4 and CD8 (FIG. 2A). FIG. 2B, both immunized and unimmunized μMT mice were left untreated, simultaneously challenged with 103 RH parasites i.p. and percent survival was measured. FIG. 2C, C57Bl/6 mice were immunized as described above, then three weeks post-final immunization whole splenocytes, CD19+ and CD8+ splenocytes were harvested and either 4×107 whole splenocytes, 1×107 CD8+ T cells, or 5×106 CD19+B cells were transferred to naïve recipient mice. Twenty-four hours after transfer mice were challenged with 103 RH parasites and monitored for survival. All experiments were performed with n=4 per group and repeated twice with similar results. The data are representative of the two experiments with similar results.

#FIG. 3 shows peritoneal excaudate inflammatory cell recruitment in response to infection with cps1-1 knock-out as compared to highly virulent strain RH. C57Bl/6 mice were infected i.p. with 1×106 cps1-1 knock-out or 1×103 RH parasites and total PECs were harvested at Days 0, 2, 4, 6, and 8. FIG. 3A, total PECs were analyzed by flow cytometry and total cell numbers recovered are presented. FIGS. 3B-3F respectively show numbers of granulocytes (GR-1+CD68+ in R3), macrophages (CD68+), inflammatory macrophages (GR-1+ CD68+), and B lymphocytes (CD19+), and T lymphocytes (CD3+) upon cps1-1 vaccination and RH infection, whereas FIGS. 3G and 3H show CD3+CD4+ and CD3+CD84+ T lymphocyte numbers upon cps1-1 vaccination (FIG. 3G) or RH infection (FIG. 3H). The data are mean absolute numbers (upper panel) or percentages of total events recorded (lower panel) and are representative of two experiments that had similar outcomes. P values are based on unpaired two tailed Students T test.

#While recombination mechanisms relying on sequence microhomology have been identified in parasites (Burton, et al. (2007) Euk. Cell 6:1773-1781), nonhomologous end-joining (NHEJ) mechanisms in parasites have not been previously described in the art. It has now been found that Toxoplasma gondii possesses NHEJ activity. By knocking out the function of the NHEJ pathway, strains exhibiting a high percentage of homologous recombination were obtained. Specifically, a strain lacking the KU80 coding region was generated. Using this ΔKu80 knockout strain, nearly 100% of transformants exhibited a double cross-over homologous recombination event resulting in gene replacement at the uracil phosphoribosyltransferase locus or the carbamoyl phosphate synthetase II locus. While NHEJ is functionally knocked out in the strains disclosed herein, with the exception of an increase in sensitivity to DNA damaging agents such as ionizing radiation and phleomycin, the strains grew normally, appeared normal, and could be maintained continuously in culture. Using the ΔKu80 knockout strain, knockout of the genes of the de novo pyrimidine synthesis pathway of T. gondii can be readily performed to generate attenuated strains of T. gondii. The attenuated strains of T. gondii elicit complete protective immunity to lethal challenge infection thereby providing a vaccine platform strategy for protecting a subject against infection by T. gondii and a non-T. gondii disease.

#Accordingly, a mutant strain of T. gondii lacking function of the KU80-dependent NHEJ is useful for the construction of gene knockouts and gene replacements in T. gondii. Such gene knockouts and gene replacements can be used in the identification of drug targets, vaccine candidates, and characterization of virulence factors. The KU80-dependent NHEJ mutant is also useful for high-throughput knockout of T. gondii genes enabling a genome-wide knockout approach to individually inactivate each of the parasite's approximately 8000 genes. The KU80-dependent NHEJ mutant is also useful for creating T. gondii strains with several or many targeted gene knockouts or other genetic alterations based on targeted double homologous recombination. KU80-dependent NHEJ mutants are useful, for example, to construct crippled strains of T. gondii that in addition to being severely attenuated in their virulence also exhibit other desirable defects such as loss of ability to develop into tissue cysts, loss of sexual stages, loss of oocyst formation, or other developmental or phenotypic defects or changes that would enhance the efficacy or safety of vaccines based on KU80-dependent NHEJ mutants. Furthermore, the KU80-dependent NHEJ mutant is useful for the identification of drug targets as well as for screening candidate drugs that have anti-parasite activity against parasites such as Plasmodium species, Cryptosporidium species, or the like. For example, the KU80-dependent NHEJ mutant enables the rapid replacement of a T. gondii gene encoding a protein of interest with the corresponding or homologous gene from another parasite (e.g., Plasmodium or Cryptosporidium), thereby enabling a high-throughput drug screen to identify inhibitors of the exogenous protein. Similarly, such strains enable the rapid design of T. gondii strains carrying and expressing foreign antigens representing components of vaccines that are capable of vaccination against T. gondii and non-T. gondii diseases. A KU80-dependent NHEJ mutant strain for the first time also enables rapid development of precise and defined genetic descriptions of T. gondii strains necessary for their potential use(s) in treating human disease(s).

#KU is known in the art as an ATP-dependent DNA helicase II protein involved in repairing DNA double-strand breaks by non-homologous end-joining (Hopfner, et al. (2002) Curr. Opin. Struct. Biol. 12:115-122; Featherstone & Jackson (1999) Mutat. Res. 434:3-15; Sandoval & Labhart (2004) DNA Repair (Amst) 3:13-21). KU is a heterodimer composed of 70 kDa and 80 kDa subunits (Walker, et al. (2001) Nature 412:607-614). Both of these subunits have strong sequence similarity and it has been suggested that they evolved by gene duplication from a homodimeric ancestor in eukaryotes (Hopfner, et al. (2002) supra). The prokaryotic KU members are homodimers and they have been predicted to be involved in the DNA repair system, which is mechanistically similar to the eukaryotic non-homologous end joining (Aravind & Koonin (2001) Genome Res. 11:1365-1374; Doherty, et al. (2001) FEBS Lett. 500:186-188). Yeast KU has been suggested to be involved in telomeric structure maintenance in addition to non-homologous end-joining (Baumann & Cech (2000) Mol. Biol. Cell 11:3265-3275). Furthermore, Neurospora crassa strains lacking KU70 and KU80 exhibit 100% homologous recombination, compared to 10 to 30% for a wild-type recipient (Ninomiya, et al. (2004) Proc. Natl. Acad. Sci. USA 101:12248-12253). Some of the phenotypes of KU-knockout mice may indicate a similar role for KU at mammalian telomeres (Yoo & Dynan (1998) Biochemistry 37:1336-1343 1998).