Live Attenuated Pru:Δcdpk2 Strain of Toxoplasma gondii Protects Against Acute, Chronic, and Congenital Toxoplasmosis

Live Attenuated Pru:Δcdpk2 Strain of Toxoplasma gondii Protects Against Acute, Chronic, and... Abstract Background The threat of Toxoplasma gondii infection in immunocompromised individuals and pregnant women necessitates the development of a safe and effective vaccine. Here, we examined the immune protection conferred by a live attenuated strain of T. gondii. Methods We tested the efficacy of intraperitoneal vaccination using 500 Ca2+-dependent protein kinase 2 (cdpk2)–deficient tachyzoites of T. gondii Pru strain against acute, chronic, and congenital toxoplasmosis in mice. The kinetics of antibody response, cytokines, and other quantifiable correlates of protection against T. gondii infection were determined. Results Vaccination with Pru:Δcdpk2 induced a high level of anti–T. gondii immunoglobulin G titer, type 1 T-helper (Th1) response at 28 days postvaccination, and a mixed Th1/type 2 T-helper response at 70 days postvaccination. All vaccinated mice survived a heterologous challenge with 1000 tachyzoites of RH or ToxoDB#9 (PYS or TgC7) strains. Also, vaccination protected against homologous infection with 20 T. gondii Pru cysts, and improved pregnancy outcome by reducing parasite cyst load in the brain, maintaining litter size and body weight of pups born to vaccinated dams challenged with 10 Pru cysts compared to pups born to unvaccinated dams. Conclusions The use of T. gondii Pru:Δcdpk2 mutant strain represents a promising approach to protection against acute, chronic, and congenital toxoplasmosis in mice. Toxoplasma gondii, vaccination, Ca2+-dependent protein kinase 2, attenuated vaccine, protective immunity Toxoplasma gondii is an obligate intracellular protozoan parasite and the causative agent of toxoplasmosis, a prevalent disease that can affect all warm-blooded animals and humans [1–3]. Infections in immunocompromised individuals, such as organ transplant recipients and AIDS patients, can cause severe or even a fatal outcome [1–3]. Primary infection during pregnancy can cause neonatal malformations, miscarriage, chorioretinitis, blindness, mental retardation, and hydrocephalus in the infected fetus [3–6]. Current treatments of toxoplasmosis are challenged by side effects of current medications [7] and the occurrence of drug-resistant strains [8]. New approaches are therefore needed to develop more effective interventions for better prevention and control of toxoplasmosis. Vaccination using live attenuated strains is promising because it can trigger a protective humoral and cellular immunity through simulating natural infection without leading to the disease [9, 10]. A commercially available vaccine, Toxovax, developed from T. gondii S48 strain, is licensed for use in sheep and goats to prevent abortion in the United Kingdom, New Zealand, France, and Ireland [11]. Attenuation of the virulence of T. gondii through deletion of certain genes has been achieved in several studies [12–18]. The Mic1-3KO RH strain, lacking mic1 and mic3 micronemal proteins, protected mice against chronic and congenital toxoplasmosis [12] and reduced T. gondii–induced abortion and tissue cyst burden in lambs born to vaccinated ewes [13]. Recently, we have shown that immunization of Kunming mice with RH:ΔGRA17 conferred significant protection against acute, chronic, and congenital T. gondii infection [14]. Other groups have shown that uracil auxotrophic mutants elicited a protective immunity against acute and chronic T. gondii infection in mice [15–18]. Toxoplasma gondii possesses 14 Ca2+-dependent protein kinases (CDPKs), which play important roles in the parasite’s motility, colonization, replication, and egress [19–23]. Recently, cdpk2 has been shown to play a role in the regulation of parasite amylopectin synthesis and degradation [23]. Disruption of cdpk2 induced abnormal accumulation of massive amylopectin granules in T. gondii tachyzoites and in the vacuolar space of the residual bodies, together with complete ablation of cyst formation [23]. Because of its inability to establish chronic infection in mice [23], Pru:Δcdpk2 mutant has emerged as an attractive candidate to produce a safe and efficacious vaccine [24]. In this study, we determined the protective efficacy of a live attenuated T. gondii Pru mutant with a target deletion of cdpk2 (designated Pru:Δcdpk2), delivered intraperitoneally. Kunming mice vaccinated with 500 Pru:Δcdpk2 tachyzoites developed a protective immune response against acute, chronic, and congenital toxoplasmosis. We also explored the possibility that if disruption of cdpk2 in RH strain impairs its virulence in mice, then a RH:Δcdpk2 mutant might be a potential vaccine candidate. MATERIALS AND METHODS Mice Eight-week-old Kunming mice were obtained from the Laboratory Animal Center of Lanzhou University. All procedures were approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Kunming mice were used in this study because of their susceptibility to acute and chronic T. gondii infection [14, 25–27] and, because of their ability to produce more pups per litter, to better assess protection against congenital infection, compared with BALB/c and C57BL/6 mice [28]. Parasites Tachyzoites of T. gondii type I (RH, PYS, and TgC7), type II (Pru), and mutant strains RH:Δcdpk2 and Pru:Δcdpk2 were maintained in human foreskin fibroblast cultures, as previously described [22, 29]. Cysts of T. gondii Pru strain were isolated from brain homogenates of Kunming mice as previously described [25–27]. Disruption of CDPK2 by CRISPR-Cas9 System The clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system was used to knockout cdpk2 gene as previously described [30]. Guide RNA and primers used in the study to generate mutant Δcdpk2 strains are listed in Supplementary Table 1. The pattern of in vitro growth of tachyzoites of T. gondii Δcdpk2 deletion mutant was compared to wild-type (WT) strain as previously described [14]. Virulence of Mutants Versus WT Strains in Mice Freshly harvested 200 tachyzoites of RH strain or RH:Δcdpk2 mutant, or 1 × 105 tachyzoites of Pru strain or Pru:Δcdpk2 mutant were injected intraperitoneally (ip) into mice (10 mice/group/parasite strain). These mice were monitored daily for the development of clinical signs of acute illness, and mortality was recorded until all mice died. Vaccination of Mice In these experiments, the efficacy of Pru:Δcdpk2 mutant strain in conferring protective immunity was evaluated against acute, chronic, and congenital T. gondii infections (Supplementary Figure 1). Mice were either vaccinated once with 500 freshly harvested Pru:Δcdpk2 tachyzoites or mock-vaccinated in a total of 200 µL phosphate-buffered saline (PBS) ip. We used the ip vaccination route because it can trigger both systemic and mucosal immune responses. Antibody Measurement Postvaccination Serum samples were obtained at 28 and 70 days postvaccination (dpv). Total immunoglobulin G (IgG) content and subclasses of IgG antibodies IgG2a and IgG1 as indicators of type 1 T-helper (Th1) and type 2 T-helper (Th2) responses, respectively, were tested by enzyme-linked immunosorbent assay (ELISA) as described previously [14, 25–27]. Vaccine-Induced Cytokine Production Mice were sacrificed 70 dpv and single splenocyte suspensions were obtained as previously described [14, 25–27]. Splenocyte cultures were incubated in vitro with 10 µg/mL T. gondii soluble tachyzoite antigen (STAg) and supernatants were tested by ELISA for the presence of secreted cytokines interleukin 2 (IL-2; 24 hours postincubation), interleukin 10 (IL-10; 72 hours postincubation), and interleukin 12 (IL-12) and interferon gamma (IFN-γ) (96 hours postincubation) in accordance with the manufacturer’s instructions (eBioscience Bender MedSystems GmbH, Austria). Protection Against Acute and Chronic Infection At 70 dpv, both vaccinated and age-matched naive mice were challenged ip with 200 μL PBS containing 1000 tachyzoites of RH, ToxoDB#9 (PYS or TgC7) strain to test efficacy against acute infection, or were inoculated orally with 20 Pru cysts to test efficacy against chronic infection. Strains of ToxoDB#9, the predominant genotype in China, have a comparable virulence to type I RH strain [31, 32]. Thus, it was sensible to determine whether immune responses induced by the Pru:Δcdpk2 mutant strain protects against infection with the local strains (TgC7 and PYS) of ToxoDB#9 genotype. It was also important to evaluate the ability of Pru:Δcdpk2 to confer protection against challenge with Pru strain, the predominant genotype during congenital infection in humans and sheep [33, 34]. The degree of illness and survival of all mice were observed daily for 35 days. Seven days postinfection (dpi), peritoneal fluid and serum samples collected from Pru:Δcdpk2-vaccinated mice infected with RH, nonvaccinated + RH-infected mice, and nonvaccinated + uninfected mice were tested for Th1 cytokines (IFN-γ and IL-12) during acute infection by ELISA. For chronic infection, mice were euthanized at 35 dpi, and their brains were removed and individually homogenized in 1 mL of PBS. The brain cyst burden was assessed by examining dilutions of Dolichos biflorus lectin-stained brain homogenates using a Zeiss wide-field epifluorescence microscope with ×10 objective, as described previously [14]. Protection Against Congenital Transmission Female mice vaccinated with 500 Pru:Δcdpk2 tachyzoites were mated with male mice 70 dpv. Two female mice were housed in a cage with 1 male, and every 12 hours female mice were inspected for the presence of vaginal plugs. The day of presence of vaginal plug was designated day 1 of gestation. On day 12 of gestation, mice were infected orally with 10 T. gondii Pru cysts. Control mouse groups included nonvaccinated uninfected mice (negative control), and nonvaccinated mice infected orally infected with 10 Pru cysts. The level of protection against congenital T. gondii infection was evaluated by analyzing litter size and survival rate of the naturally delivered pups at birth and 35 days old. The body weight of pups at 35 days old was also used to evaluate the protective efficacy. The level of maternal protection in Pru cyst–challenged dams and their pups was determined by quantifying the brain tissue cyst burden in surviving pups at 35 days of age, and in their dams at 30 days after delivery. Maternal splenocytes from pregnant mice challenged with 10 Pru cysts on day 12 of gestation were collected 6 days later. Splenocyte culture was stimulated with 10 µg/mL STAg and the level of Th1 (IFN-γ, IL-2, IL-12), and Th2 (IL-10) cytokines in vaccinated + infected mice, nonvaccinated + infected mice, and nonvaccinated + uninfected mice was evaluated using ELISA, as described above. Statistical Analysis The differences in the level of cytokines, anti–T. gondii antibodies and parasite cysts’ burdens were compared using 2-tailed, unpaired Student t test (for comparing means between 2 groups) or 1-way analysis of variance (for comparing means between ≥3 groups). The standard deviation was derived from 3 independently performed experiments with 3 replicates per experiment for the in vitro assays. Values of P < .05 were considered statistically significant. Mortality was determined by plotting survival curves of the different mouse groups stratified by T. gondii infection and vaccination status by Mantel–Cox log-rank test. RESULTS Deletion of cdpk2 Gene in T. gondii RH and Pru Strains The cdpk2 gene was successfully knocked out in RH and Pru strains using CRISPR-Cas9 system. In both strains, the DHFR* was inserted into the guide RNA–targeted coding sequence region through nonhomologous end joining (Supplementary Figure 2A). Single, stable pyrimethamine-resistant clones were generated and verified by specific polymerase chain reaction method (Supplementary Figure 2B). Phenotypic characterization of RH:Δcdpk2 or Pru:Δcdpk2 tachyzoites revealed abnormal morphology and excessive accumulation of granules at both the tachyzoites’ basal end and within the tachyzoites’ residual body, whereas tachyzoites of the WT RH and Pru strains appeared normal (Supplementary Figure 3). These results demonstrate that cdpk2 gene was successfully knocked out in the mutant RH and Pru strains. Disruption of cdpk2 Does Not Affect Virulence in Mice Deletion of cdpk2 gene did not result in attenuation of the virulence of mutant RH or Pru strains, as indicated by the comparable survival rates of mice infected with the parental WT or mutant strains (Supplementary Figure 4). In an effort to achieve the required level of immunogenicity of the attenuated Pru:Δcdpk2 strain, without inducing an excessive immune response or death in the vaccinated mice, we reduced the number of type II Pru:Δcdpk2 tachyzoites used in the vaccination experiments from 105 to 500 tachyzoites. Mice challenged with 500 Pru:Δcdpk2 mutant tachyzoites survived and consistent with previous work [23], tissue cysts were not detected in the brain of mice challenged with 500 Pru:Δcdpk2 tachyzoites, but brain tissue cysts were detected in mice challenged with 500 WT Pru tachyzoites (data not shown). Humoral Immune Responses Induced by Vaccination The immunogenicity of Pru:Δcdpk2 was assessed by determination of specific anti–T. gondii IgG antibody titers and IgG isotypes in the serum of vaccinated mice at 28 and 70 dpv by quantitative ELISA. At day 28 after vaccination, all vaccinated mice had seroconverted with a higher level of anti–T. gondii IgG antibodies compared with nonvaccinated mice. This level of IgG titer remained high at 70 dpv (Figure 1). These results suggest that Pru:Δcdpk2 induced a strong humoral response. We then tested whether a Th1 and Th2 response was elicited in the vaccinated mice by evaluating the levels of STAg-specific IgG subclasses (IgG2a and IgG1 isotypes, respectively). Compared to nonvaccinated mice, the level of IgG2a was significantly higher in vaccinated mice at 28 and 70 dpv. The level of IgG1 was only increased in the vaccinated mice at 70 dpv, compared with nonvaccinated mice (Figure 1). These results suggest that vaccination with Pru:Δcdpk2 in mice elicits a Th1-type immune response at 28 dpv and a mixed Th1/Th2 immune response at 70 dpv. Figure 1. View largeDownload slide Humoral response and antibody isotype profile in the serum of mice vaccinated with Pru:Δcdpk2 by intraperitoneal route. Levels of immunoglobulin G (IgG) and IgG subclass (IgG2a and IgG1) antibodies were evaluated in the sera of mice at 28 and 70 days after Pru:Δcdpk2 vaccination compared to nonvaccinated mice. The patterns of IgG2a to IgG1 show the induction of a type 1 T-helper (Th1) immune response at day 28 postvaccination, followed by a mixed Th1/type 2 T-helper immune response at day 70 postvaccination. Results are expressed as mean of optical density (OD) 450 mm ± standard deviation. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Figure 1. View largeDownload slide Humoral response and antibody isotype profile in the serum of mice vaccinated with Pru:Δcdpk2 by intraperitoneal route. Levels of immunoglobulin G (IgG) and IgG subclass (IgG2a and IgG1) antibodies were evaluated in the sera of mice at 28 and 70 days after Pru:Δcdpk2 vaccination compared to nonvaccinated mice. The patterns of IgG2a to IgG1 show the induction of a type 1 T-helper (Th1) immune response at day 28 postvaccination, followed by a mixed Th1/type 2 T-helper immune response at day 70 postvaccination. Results are expressed as mean of optical density (OD) 450 mm ± standard deviation. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Cytokine Production After Vaccination Cytokine production in splenocyte culture supernatants stimulated with STAg was assessed by ELISA at 70 dpv. The levels of Th1-type cytokines (IFN-γ, IL-2, and IL-12) were significantly higher than those in nonvaccinated mice. Also, the level of Th2-type cytokine (IL-10) was significantly higher than that of nonvaccinated mice (Figure 2). Figure 2. View largeDownload slide Levels of type 1 T-helper (Th1) and type 2 T-helper (Th2) cytokines produced by splenocytes culture of Pru:Δcdpk2-vaccinated mice. Spleen cells from 6 mice were obtained 70 days postvaccination and stimulated in vitro with 10 µg/mL soluble Toxoplasma gondii tachyzoite antigen. Cell-free supernatants were harvested and evaluated for Th1 (interferon gamma [IFN-γ], interleukin 2 [IL-2], and interleukin 12 [IL-12]) and Th2 (interleukin 10 [IL-10]) cytokines using enzyme-linked immunosorbent assay. Significance compared with control (uninfected + nonvaccinated) mice: **P < .01, ***P < .001. Figure 2. View largeDownload slide Levels of type 1 T-helper (Th1) and type 2 T-helper (Th2) cytokines produced by splenocytes culture of Pru:Δcdpk2-vaccinated mice. Spleen cells from 6 mice were obtained 70 days postvaccination and stimulated in vitro with 10 µg/mL soluble Toxoplasma gondii tachyzoite antigen. Cell-free supernatants were harvested and evaluated for Th1 (interferon gamma [IFN-γ], interleukin 2 [IL-2], and interleukin 12 [IL-12]) and Th2 (interleukin 10 [IL-10]) cytokines using enzyme-linked immunosorbent assay. Significance compared with control (uninfected + nonvaccinated) mice: **P < .01, ***P < .001. Pru:Δcdpk2 Immunogenicity and Potency for Protection Against Acute Infection Kunming mice were vaccinated with 500 Pru:Δcdpk2 tachyzoites and, 70 days later, challenged with 1000 tachyzoites of T. gondii type I RH strain, or ToxoDB#9 (PYS or TgC7) strain. As expected, all nonvaccinated mice infected with 1000 RH strain or ToxoDB#9 (PYS or TgC7) strains died within 10 days after infection, whereas all mice vaccinated survived regardless of the challenging strains (Figure 3). We further analyzed Th1 cytokines (IL-12 and IFN-γ) in the serum and peritoneal fluids at 7 days after infection. Significantly elevated levels of IL-12 and IFN-γ were found in the nonvaccinated + RH-infected mice, whereas only modestly elevated levels of the same cytokines was observed in Pru:Δcdpk2 vaccinated + RH-infected mice (Figure 4). Figure 3. View largeDownload slide Protection of mice against acute Toxoplasma gondii infection. Survival curves of Pru:Δcdpk2-vaccinated mice, challenged intraperitoneally with 103 tachyzoites of RH, PYS, or TgC7 strains 70 days postvaccination. The survival of mice was monitored for 35 days. A log-rank (Mantel–Cox) test showed significant difference in the survival rates between vaccinated + infected groups compared to nonvaccinated + infected groups (P = .003). All mice in the vaccinated groups remained alive at day 35 after infection, but all mice in the nonvaccinated groups died between day 7 and 10 after infection. In addition to the separate curves that show the difference in the survival rate between vaccinated and nonvaccinated mouse groups infected with RH, PYS, or TgC7 strains (A–C), the overall survival rates in all vaccinated mouse groups compared with the nonvaccinated mouse groups were combined in 1 graph (D). Figure 3. View largeDownload slide Protection of mice against acute Toxoplasma gondii infection. Survival curves of Pru:Δcdpk2-vaccinated mice, challenged intraperitoneally with 103 tachyzoites of RH, PYS, or TgC7 strains 70 days postvaccination. The survival of mice was monitored for 35 days. A log-rank (Mantel–Cox) test showed significant difference in the survival rates between vaccinated + infected groups compared to nonvaccinated + infected groups (P = .003). All mice in the vaccinated groups remained alive at day 35 after infection, but all mice in the nonvaccinated groups died between day 7 and 10 after infection. In addition to the separate curves that show the difference in the survival rate between vaccinated and nonvaccinated mouse groups infected with RH, PYS, or TgC7 strains (A–C), the overall survival rates in all vaccinated mouse groups compared with the nonvaccinated mouse groups were combined in 1 graph (D). Figure 4. View largeDownload slide Proinflammatory cytokines produced by Pru:Δcdpk2-vaccinated mice after infection with Toxoplasma gondii RH. Mice were challenged with 103 RH tachyzoites 70 days postvaccination and levels of interferon gamma (IFN-γ) and interleukin 12 (IL-12) in the serum and peritoneal washes were assessed by enzyme-linked immunosorbent assay at 7 days postinfection. Highest levels of proinflammatory cytokines were found in the nonvaccinated infected mice. Significance compared with uninfected and nonvaccinated control mice: **P < .01, ***P < .001. Figure 4. View largeDownload slide Proinflammatory cytokines produced by Pru:Δcdpk2-vaccinated mice after infection with Toxoplasma gondii RH. Mice were challenged with 103 RH tachyzoites 70 days postvaccination and levels of interferon gamma (IFN-γ) and interleukin 12 (IL-12) in the serum and peritoneal washes were assessed by enzyme-linked immunosorbent assay at 7 days postinfection. Highest levels of proinflammatory cytokines were found in the nonvaccinated infected mice. Significance compared with uninfected and nonvaccinated control mice: **P < .01, ***P < .001. Protection Against Chronic Infection The protective efficacy of vaccination with Pru:Δcdpk2 against chronic infection was investigated in Kunming mice at 70 dpv. All Pru:Δcdpk2-vaccinated mice survived infection with 20 T. gondii Pru parasite cysts, whereas only 40% of nonvaccinated infected mice survived (Figure 5A). At 35 days postchallenge, parasite cyst burden in the brain of surviving Pru:Δcdpk2-vaccinated compared with nonvaccinated mice was determined. Nonvaccinated mice challenged with 20 Pru cysts had 4296 ± 687 cysts per brain, whereas Pru:Δcdpk2-vaccinated mice challenged with the same number of Pru cysts had significantly fewer cysts per brain (78 ± 48 cysts/brain) (P < .001; Figure 5B). Figure 5. View largeDownload slide Vaccination with Pru:Δcdpk2 promoted survival and reduced brain cyst’s burden after infection. A, Survival curves following challenge of Pru:Δcdpk2-vaccinated mice with 20 cysts of Toxoplasma gondii Pru strain, compared with nonvaccinated mice, 70 days postvaccination. The survival of mice was monitored for 35 days. A log-rank (Mantel–Cox) test demonstrated a significant difference between the vaccinated and nonvaccinated groups (P = .0001). B, Cyst burden in the brain of mice that survived to 35 days after challenge (Pru:Δcdpk2-vaccinated + Pru-infected mice vs nonvaccinated + Pru-infected mice). Data points indicate mean ± SD. ***P < .001. Figure 5. View largeDownload slide Vaccination with Pru:Δcdpk2 promoted survival and reduced brain cyst’s burden after infection. A, Survival curves following challenge of Pru:Δcdpk2-vaccinated mice with 20 cysts of Toxoplasma gondii Pru strain, compared with nonvaccinated mice, 70 days postvaccination. The survival of mice was monitored for 35 days. A log-rank (Mantel–Cox) test demonstrated a significant difference between the vaccinated and nonvaccinated groups (P = .0001). B, Cyst burden in the brain of mice that survived to 35 days after challenge (Pru:Δcdpk2-vaccinated + Pru-infected mice vs nonvaccinated + Pru-infected mice). Data points indicate mean ± SD. ***P < .001. Protection Against Congenital Toxoplasmosis Pregnant mice were orally challenged with 10 T. gondii Pru cysts on day 12 of gestation, and the litter size and body weight of the neonates were determined. The cyst burden in the brain of neonates and dams was evaluated. The litter size and survival of pups of Pru:Δcdpk2-vaccinated + Pru cyst–infected dams was similar to that of nonvaccinated uninfected mice (Figure 6A). Body weight of neonates born to Pru:Δcdpk2-vaccinated + Pru cyst–infected dams was similar to neonates born to nonvaccinated uninfected dams (Figure 6B). In contrast, the litter size and body weight of pups of nonvaccinated + Pru cyst–infected mice were significantly lower compared with those of nonvaccinated uninfected mice or Pru:Δcdpk2-vaccinated + Pru cyst–infected mice. The brain cyst burden in the neonates was determined at day 35 postpartum. The average brain cyst number in all neonates (n = 19) born to nonvaccinated + Pru cyst–infected dams was 919 ± 339. In contrast, 41.4% (24/58) of neonates born to Pru:Δcdpk2-vaccinated + Pru cyst–infected dams had an average brain cyst number of (60 ± 33). Interestingly, examination of the brain of 58.6% (34/58) of neonates born to vaccinated + infected dams revealed no cysts. The brain cyst burden in dams was also determined at day 30 postpartum. The average brain cyst number was significantly higher in nonvaccinated + Pru cyst–infected dams (3287 ± 569 cysts/brain) than in that of Pru:Δcdpk2-vaccinated + Pru cyst–infected dams (77 ± 58 cysts/ brain). Figure 6. View largeDownload slide Protection of mice against type II Pru cyst infection on day 12 of gestation. A, Litter size and survival of pups from nonvaccinated uninfected mice, nonvaccinated + Pru-infected mice, and Pru:Δcdpk2-vaccinated + Pru-infected mice was assessed at birth and 35 days after birth. B, The average body weight of 35-day-old pups. Data points indicate mean ± standard deviation. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Figure 6. View largeDownload slide Protection of mice against type II Pru cyst infection on day 12 of gestation. A, Litter size and survival of pups from nonvaccinated uninfected mice, nonvaccinated + Pru-infected mice, and Pru:Δcdpk2-vaccinated + Pru-infected mice was assessed at birth and 35 days after birth. B, The average body weight of 35-day-old pups. Data points indicate mean ± standard deviation. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Immune Responses to Infection During Pregnancy The levels of T. gondii–specific Th1 (IFN-γ, IL-12, IL-2) and Th2 (IL-10) cytokines from Pru:Δcdpk2-vaccinated + Pru cyst–infected mice were significantly higher compared with those in nonvaccinated + Pru cyst–infected mice and nonvaccinated uninfected mice (P < .001; Figure 7). However, the difference in the cytokine levels between nonvaccinated + Pru cyst–infected and nonvaccinated uninfected mice was not statistically significant (P > .05). Figure 7. View largeDownload slide Profile of Toxoplasma gondii–specific type 1 T-helper (Th1) and type 2 T-helper (Th2) cytokines in Pru:Δcdpk2-vaccinated mice after infection with Pru cysts. Vaccinated, pregnant mice were infected with T. gondii Pru cysts at 12 days of gestation and spleen cells were collected 6 days after infection. Splenocyte culture was stimulated with 10 µg/mL T. gondii soluble tachyzoite antigen. Cell-free supernatants were evaluated for Th1 (interferon gamma [IFN-γ], interleukin 2 [IL-2], and interleukin 12 [IL-12]) and Th2 (interleukin 10 [IL-10]). Cytokine concentrations represent mean ± standard deviation after subtraction of background control values with medium only. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Figure 7. View largeDownload slide Profile of Toxoplasma gondii–specific type 1 T-helper (Th1) and type 2 T-helper (Th2) cytokines in Pru:Δcdpk2-vaccinated mice after infection with Pru cysts. Vaccinated, pregnant mice were infected with T. gondii Pru cysts at 12 days of gestation and spleen cells were collected 6 days after infection. Splenocyte culture was stimulated with 10 µg/mL T. gondii soluble tachyzoite antigen. Cell-free supernatants were evaluated for Th1 (interferon gamma [IFN-γ], interleukin 2 [IL-2], and interleukin 12 [IL-12]) and Th2 (interleukin 10 [IL-10]). Cytokine concentrations represent mean ± standard deviation after subtraction of background control values with medium only. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. DISCUSSION A highly effective vaccine against toxoplasmosis is urgently required, with leading vaccination strategies targeting both horizontal transmission and vertical transmission from pregnant dams to their offspring. Previous study has shown that cdpk2 is essential for the development of viable T. gondii tissue cysts, as cdpk2-deficient parasite strains failed to form cysts in the brain of mice [23]. Based on this observation, we inferred that cdpk2-deficient T. gondii might be a promising live attenuated vaccine to prevent toxoplasmosis. In the present study, cdpk2 was successfully disrupted in T. gondii RH and Pru strains using CRISPR-Cas9 method; the mutant strains exhibited the anticipated morphological abnormalities, and no parasite cyst was detected in the brain of mice challenged with Pru:Δcdpk2 tachyzoites, which was consistent with previous study [23]. Our results showed that deletion of the cdpk2 gene did not result in the attenuation of the virulence of RH or Pru strains, as indicated by the comparable survival rates of mice infected with the parental WT (RH or Pru) or mutant (RH:Δcdpk2 or Pru:Δcdpk2) strains. A high dose (105) of Pru:Δcdpk2 tachyzoites caused mice death in 11 days; however, all mice infected with the same strain, but with a smaller dose (500), survived and no parasite cyst was detected in their brain. Therefore, 500 Pru:Δcdpk2 tachyzoites were used in all subsequent experiments as the vaccination dose because it exhibited the type of balance between safety and high immunogenicity that should be expected of a promising live attenuated vaccine strain. Consistent with other attenuated T. gondii vaccines, vaccination with Pru:Δcdpk2 induced a high level of anti–T. gondii IgG antibodies in mice [12–18]. These specific IgG antibodies can neutralize the attachment of T. gondii and limit its ability to establish infection [35–38]. Pru:Δcdpk2-vaccinated mice also developed a sequential Th1 and a mixed Th1/Th2-type immune response, as indicated by the high level of Th1 immunity (IgG2a) at day 28 postvaccination; and the elevation of Th2 protective (IgG1) level at day 70 dpv when compared to that in nonvaccinated mice. The high level of IgG2a and IgG1 in Pru:Δcdpk2-vaccinated mice was substantiated by high levels of Th1 cytokines (IFN-γ, IL-2, and IL-12) and Th2 cytokines (IL-10) at 70 dpv. Indeed, the immune response induced by Pru:Δcdpk2 vaccination completely protected mice (ie, 100% survival rate) postchallenge with RH, or ToxoDB#9 (PYS or TgC7) strain, whereas nonvaccinated mice died within 10 dpi. In addition, Pru:Δcdpk2 significantly reduced the development of chronic toxoplasmosis. The parasite cysts were significantly reduced in the brain of vaccinated mice after infection with 20 cysts of T. gondii Pru strain compared with nonvaccinated + Pru cyst–infected mice. In our study, Th1 cytokines from the nonvaccinated + RH-infected mice were significantly higher than those from the Pru:Δcdpk2 vaccinated + RH-infected mice, indicating that vaccination with Pru:Δcdpk2 may have down-regulated Th1 response and reduced the severe inflammatory response that accompanies acute T. gondii infection [39–42]. Th1-type cytokine responses with high levels of IFN-γ, IL-2, and IL-12 are needed to limit congenital transmission and to protect pregnant mice against T. gondii infection [43–46]. However, Th1-type immune response can be detrimental to pregnancy and potentially deleterious for the conceptus [43, 44]. Thus, a balanced Th1/Th2 immune response detected at day 70 postvaccination seems to contribute to the observed successful pregnancy outcomes, as unsuccessful pregnancies were observed at day 28 postvaccination (data not shown) where Th1 response was dominating (Figure 1). Therefore, the high level of the Th2-related anti-inflammatory cytokine IL-10, which neutralizes the production of Th1 cytokines (IFN-γ and IL-12) detected in Pru:Δcdpk2-vaccinated pregnant mice infected with Pru cysts on day 12 of gestation (Figure 7) may have contributed to better pregnancy outcomes compared with nonvaccinated + Pru-infected pregnant mice. Higher IL-10 levels appear to play a role in protection against infection, because IL-10–deficient mice died during acute T. gondii infection due to a strong Th1 inflammatory response [47]. Previous studies showed that pregnancy outcomes can be improved in T. gondii–infected mice by administration of recombinant IL-10 and can be worsened in IL-10–deficient mice [48]. Despite the inhibitory effect of maternal and fetal Th2 polarized immune response that occurs during pregnancy on Th1 cytokine production, vaccination seems to elicit a protective immune response that enabled the pregnant mice to control the infection. These results show that vaccination of mice with Pru:Δcdpk2 achieved a critical balance between Th1 and Th2 responses for optimal control of infection, while minimizing overt inflammation and severe pathology. Although the immune responses induced by Pru:Δcdpk2 vaccination cannot completely block the vertical transmission, a statistically lower brain cyst burden was observed in pups from Pru:Δcdpk2-vaccinated + Pru-infected dams, compared to pups of nonvaccinated + Pru-infected dams. In addition, there were significantly fewer numbers of brain cysts in Pru:Δcdpk2-vaccinated + Pru-infected dams than in nonvaccinated + Pru-infected dams. Also, neonates born to Pru:Δcdpk2-vaccinated + Pru-infected dams had similar body weights to neonates born to nonvaccinated + uninfected mice. These results indicate that vaccination with Pru:Δcdpk2 can confer significant protection against subsequent oral infection with Pru cysts during the second trimester of gestation. Unexpectedly, infection with T. gondii Pru strain at 12 days of gestation failed to elicit more cytokines in nonvaccinated + Pru-infected mice, compared with nonvaccinated + uninfected mice (Figure 7). At present, the reason for this observation is unknown. However, it is possible that the time elapsed after infection was not long enough to elicit a detectable level of cytokines nor to induce potent CD8+ T-cell responses without prior antigenic stimulation. In conclusion, data reported in this study demonstrate that a single ip vaccination of 500 Pru:Δcdpk2 tachyzoites provides cross-protection against homologous and heterologous challenge with multiple T. gondii strains of the same and different genotypes in mice. Pru:Δcdpk2 strain generated an immune response, which improved the survival rate and reduced parasite cyst burden in the brain of the vaccinated mice. Strong protection against congenital toxoplasmosis was demonstrated by a significant reduction in the brain tissue cyst burden in the pups and their dams, along with improvement in the body weight and survival rate of pups born to vaccinated dams compared with nonvaccinated dams. Given the potential efficacy of Pru:Δcdpk2 live attenuated vaccine, the data generated in Kunming mice merit further exploration and should be evaluated in larger animals, such as sheep. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. The authors thank Luke Matthews for contributing to the design of the graphs and Paul Goodwin for critical comments on the manuscript. Author contributions. X. Q. Z., S. Y. H., and H. M. E. conceived the project, designed the experiments, and critically revised the manuscript. J. L. W., T. T. L., and K. C. performed the experiments and analyzed the data. J. L. W. drafted the manuscript. W. C., W. B. Y., and M. J. B. helped in the implementation of the study. All authors reviewed and approved the final version of the manuscript. Financial support. This research was supported by the National Natural Science Foundation of China (grant numbers 31472184 and 31230073); the Natural Science Foundation of Gansu Province for Distinguished Young Scholars (grant number 1506RJDA133); the National Key Research and Development Program of China (grant number 2017YFD0501304); and the Elite Program of Chinese Academy of Agricultural Sciences. Potential conflicts of interest. All authors: No potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet  2004; 363: 1965– 76. Google Scholar CrossRef Search ADS PubMed  2. Robert-Gangneux F, Belaz S. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Live Attenuated Pru:Δcdpk2 Strain of Toxoplasma gondii Protects Against Acute, Chronic, and Congenital Toxoplasmosis

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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1537-6613
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10.1093/infdis/jiy211
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Abstract

Abstract Background The threat of Toxoplasma gondii infection in immunocompromised individuals and pregnant women necessitates the development of a safe and effective vaccine. Here, we examined the immune protection conferred by a live attenuated strain of T. gondii. Methods We tested the efficacy of intraperitoneal vaccination using 500 Ca2+-dependent protein kinase 2 (cdpk2)–deficient tachyzoites of T. gondii Pru strain against acute, chronic, and congenital toxoplasmosis in mice. The kinetics of antibody response, cytokines, and other quantifiable correlates of protection against T. gondii infection were determined. Results Vaccination with Pru:Δcdpk2 induced a high level of anti–T. gondii immunoglobulin G titer, type 1 T-helper (Th1) response at 28 days postvaccination, and a mixed Th1/type 2 T-helper response at 70 days postvaccination. All vaccinated mice survived a heterologous challenge with 1000 tachyzoites of RH or ToxoDB#9 (PYS or TgC7) strains. Also, vaccination protected against homologous infection with 20 T. gondii Pru cysts, and improved pregnancy outcome by reducing parasite cyst load in the brain, maintaining litter size and body weight of pups born to vaccinated dams challenged with 10 Pru cysts compared to pups born to unvaccinated dams. Conclusions The use of T. gondii Pru:Δcdpk2 mutant strain represents a promising approach to protection against acute, chronic, and congenital toxoplasmosis in mice. Toxoplasma gondii, vaccination, Ca2+-dependent protein kinase 2, attenuated vaccine, protective immunity Toxoplasma gondii is an obligate intracellular protozoan parasite and the causative agent of toxoplasmosis, a prevalent disease that can affect all warm-blooded animals and humans [1–3]. Infections in immunocompromised individuals, such as organ transplant recipients and AIDS patients, can cause severe or even a fatal outcome [1–3]. Primary infection during pregnancy can cause neonatal malformations, miscarriage, chorioretinitis, blindness, mental retardation, and hydrocephalus in the infected fetus [3–6]. Current treatments of toxoplasmosis are challenged by side effects of current medications [7] and the occurrence of drug-resistant strains [8]. New approaches are therefore needed to develop more effective interventions for better prevention and control of toxoplasmosis. Vaccination using live attenuated strains is promising because it can trigger a protective humoral and cellular immunity through simulating natural infection without leading to the disease [9, 10]. A commercially available vaccine, Toxovax, developed from T. gondii S48 strain, is licensed for use in sheep and goats to prevent abortion in the United Kingdom, New Zealand, France, and Ireland [11]. Attenuation of the virulence of T. gondii through deletion of certain genes has been achieved in several studies [12–18]. The Mic1-3KO RH strain, lacking mic1 and mic3 micronemal proteins, protected mice against chronic and congenital toxoplasmosis [12] and reduced T. gondii–induced abortion and tissue cyst burden in lambs born to vaccinated ewes [13]. Recently, we have shown that immunization of Kunming mice with RH:ΔGRA17 conferred significant protection against acute, chronic, and congenital T. gondii infection [14]. Other groups have shown that uracil auxotrophic mutants elicited a protective immunity against acute and chronic T. gondii infection in mice [15–18]. Toxoplasma gondii possesses 14 Ca2+-dependent protein kinases (CDPKs), which play important roles in the parasite’s motility, colonization, replication, and egress [19–23]. Recently, cdpk2 has been shown to play a role in the regulation of parasite amylopectin synthesis and degradation [23]. Disruption of cdpk2 induced abnormal accumulation of massive amylopectin granules in T. gondii tachyzoites and in the vacuolar space of the residual bodies, together with complete ablation of cyst formation [23]. Because of its inability to establish chronic infection in mice [23], Pru:Δcdpk2 mutant has emerged as an attractive candidate to produce a safe and efficacious vaccine [24]. In this study, we determined the protective efficacy of a live attenuated T. gondii Pru mutant with a target deletion of cdpk2 (designated Pru:Δcdpk2), delivered intraperitoneally. Kunming mice vaccinated with 500 Pru:Δcdpk2 tachyzoites developed a protective immune response against acute, chronic, and congenital toxoplasmosis. We also explored the possibility that if disruption of cdpk2 in RH strain impairs its virulence in mice, then a RH:Δcdpk2 mutant might be a potential vaccine candidate. MATERIALS AND METHODS Mice Eight-week-old Kunming mice were obtained from the Laboratory Animal Center of Lanzhou University. All procedures were approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Kunming mice were used in this study because of their susceptibility to acute and chronic T. gondii infection [14, 25–27] and, because of their ability to produce more pups per litter, to better assess protection against congenital infection, compared with BALB/c and C57BL/6 mice [28]. Parasites Tachyzoites of T. gondii type I (RH, PYS, and TgC7), type II (Pru), and mutant strains RH:Δcdpk2 and Pru:Δcdpk2 were maintained in human foreskin fibroblast cultures, as previously described [22, 29]. Cysts of T. gondii Pru strain were isolated from brain homogenates of Kunming mice as previously described [25–27]. Disruption of CDPK2 by CRISPR-Cas9 System The clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system was used to knockout cdpk2 gene as previously described [30]. Guide RNA and primers used in the study to generate mutant Δcdpk2 strains are listed in Supplementary Table 1. The pattern of in vitro growth of tachyzoites of T. gondii Δcdpk2 deletion mutant was compared to wild-type (WT) strain as previously described [14]. Virulence of Mutants Versus WT Strains in Mice Freshly harvested 200 tachyzoites of RH strain or RH:Δcdpk2 mutant, or 1 × 105 tachyzoites of Pru strain or Pru:Δcdpk2 mutant were injected intraperitoneally (ip) into mice (10 mice/group/parasite strain). These mice were monitored daily for the development of clinical signs of acute illness, and mortality was recorded until all mice died. Vaccination of Mice In these experiments, the efficacy of Pru:Δcdpk2 mutant strain in conferring protective immunity was evaluated against acute, chronic, and congenital T. gondii infections (Supplementary Figure 1). Mice were either vaccinated once with 500 freshly harvested Pru:Δcdpk2 tachyzoites or mock-vaccinated in a total of 200 µL phosphate-buffered saline (PBS) ip. We used the ip vaccination route because it can trigger both systemic and mucosal immune responses. Antibody Measurement Postvaccination Serum samples were obtained at 28 and 70 days postvaccination (dpv). Total immunoglobulin G (IgG) content and subclasses of IgG antibodies IgG2a and IgG1 as indicators of type 1 T-helper (Th1) and type 2 T-helper (Th2) responses, respectively, were tested by enzyme-linked immunosorbent assay (ELISA) as described previously [14, 25–27]. Vaccine-Induced Cytokine Production Mice were sacrificed 70 dpv and single splenocyte suspensions were obtained as previously described [14, 25–27]. Splenocyte cultures were incubated in vitro with 10 µg/mL T. gondii soluble tachyzoite antigen (STAg) and supernatants were tested by ELISA for the presence of secreted cytokines interleukin 2 (IL-2; 24 hours postincubation), interleukin 10 (IL-10; 72 hours postincubation), and interleukin 12 (IL-12) and interferon gamma (IFN-γ) (96 hours postincubation) in accordance with the manufacturer’s instructions (eBioscience Bender MedSystems GmbH, Austria). Protection Against Acute and Chronic Infection At 70 dpv, both vaccinated and age-matched naive mice were challenged ip with 200 μL PBS containing 1000 tachyzoites of RH, ToxoDB#9 (PYS or TgC7) strain to test efficacy against acute infection, or were inoculated orally with 20 Pru cysts to test efficacy against chronic infection. Strains of ToxoDB#9, the predominant genotype in China, have a comparable virulence to type I RH strain [31, 32]. Thus, it was sensible to determine whether immune responses induced by the Pru:Δcdpk2 mutant strain protects against infection with the local strains (TgC7 and PYS) of ToxoDB#9 genotype. It was also important to evaluate the ability of Pru:Δcdpk2 to confer protection against challenge with Pru strain, the predominant genotype during congenital infection in humans and sheep [33, 34]. The degree of illness and survival of all mice were observed daily for 35 days. Seven days postinfection (dpi), peritoneal fluid and serum samples collected from Pru:Δcdpk2-vaccinated mice infected with RH, nonvaccinated + RH-infected mice, and nonvaccinated + uninfected mice were tested for Th1 cytokines (IFN-γ and IL-12) during acute infection by ELISA. For chronic infection, mice were euthanized at 35 dpi, and their brains were removed and individually homogenized in 1 mL of PBS. The brain cyst burden was assessed by examining dilutions of Dolichos biflorus lectin-stained brain homogenates using a Zeiss wide-field epifluorescence microscope with ×10 objective, as described previously [14]. Protection Against Congenital Transmission Female mice vaccinated with 500 Pru:Δcdpk2 tachyzoites were mated with male mice 70 dpv. Two female mice were housed in a cage with 1 male, and every 12 hours female mice were inspected for the presence of vaginal plugs. The day of presence of vaginal plug was designated day 1 of gestation. On day 12 of gestation, mice were infected orally with 10 T. gondii Pru cysts. Control mouse groups included nonvaccinated uninfected mice (negative control), and nonvaccinated mice infected orally infected with 10 Pru cysts. The level of protection against congenital T. gondii infection was evaluated by analyzing litter size and survival rate of the naturally delivered pups at birth and 35 days old. The body weight of pups at 35 days old was also used to evaluate the protective efficacy. The level of maternal protection in Pru cyst–challenged dams and their pups was determined by quantifying the brain tissue cyst burden in surviving pups at 35 days of age, and in their dams at 30 days after delivery. Maternal splenocytes from pregnant mice challenged with 10 Pru cysts on day 12 of gestation were collected 6 days later. Splenocyte culture was stimulated with 10 µg/mL STAg and the level of Th1 (IFN-γ, IL-2, IL-12), and Th2 (IL-10) cytokines in vaccinated + infected mice, nonvaccinated + infected mice, and nonvaccinated + uninfected mice was evaluated using ELISA, as described above. Statistical Analysis The differences in the level of cytokines, anti–T. gondii antibodies and parasite cysts’ burdens were compared using 2-tailed, unpaired Student t test (for comparing means between 2 groups) or 1-way analysis of variance (for comparing means between ≥3 groups). The standard deviation was derived from 3 independently performed experiments with 3 replicates per experiment for the in vitro assays. Values of P < .05 were considered statistically significant. Mortality was determined by plotting survival curves of the different mouse groups stratified by T. gondii infection and vaccination status by Mantel–Cox log-rank test. RESULTS Deletion of cdpk2 Gene in T. gondii RH and Pru Strains The cdpk2 gene was successfully knocked out in RH and Pru strains using CRISPR-Cas9 system. In both strains, the DHFR* was inserted into the guide RNA–targeted coding sequence region through nonhomologous end joining (Supplementary Figure 2A). Single, stable pyrimethamine-resistant clones were generated and verified by specific polymerase chain reaction method (Supplementary Figure 2B). Phenotypic characterization of RH:Δcdpk2 or Pru:Δcdpk2 tachyzoites revealed abnormal morphology and excessive accumulation of granules at both the tachyzoites’ basal end and within the tachyzoites’ residual body, whereas tachyzoites of the WT RH and Pru strains appeared normal (Supplementary Figure 3). These results demonstrate that cdpk2 gene was successfully knocked out in the mutant RH and Pru strains. Disruption of cdpk2 Does Not Affect Virulence in Mice Deletion of cdpk2 gene did not result in attenuation of the virulence of mutant RH or Pru strains, as indicated by the comparable survival rates of mice infected with the parental WT or mutant strains (Supplementary Figure 4). In an effort to achieve the required level of immunogenicity of the attenuated Pru:Δcdpk2 strain, without inducing an excessive immune response or death in the vaccinated mice, we reduced the number of type II Pru:Δcdpk2 tachyzoites used in the vaccination experiments from 105 to 500 tachyzoites. Mice challenged with 500 Pru:Δcdpk2 mutant tachyzoites survived and consistent with previous work [23], tissue cysts were not detected in the brain of mice challenged with 500 Pru:Δcdpk2 tachyzoites, but brain tissue cysts were detected in mice challenged with 500 WT Pru tachyzoites (data not shown). Humoral Immune Responses Induced by Vaccination The immunogenicity of Pru:Δcdpk2 was assessed by determination of specific anti–T. gondii IgG antibody titers and IgG isotypes in the serum of vaccinated mice at 28 and 70 dpv by quantitative ELISA. At day 28 after vaccination, all vaccinated mice had seroconverted with a higher level of anti–T. gondii IgG antibodies compared with nonvaccinated mice. This level of IgG titer remained high at 70 dpv (Figure 1). These results suggest that Pru:Δcdpk2 induced a strong humoral response. We then tested whether a Th1 and Th2 response was elicited in the vaccinated mice by evaluating the levels of STAg-specific IgG subclasses (IgG2a and IgG1 isotypes, respectively). Compared to nonvaccinated mice, the level of IgG2a was significantly higher in vaccinated mice at 28 and 70 dpv. The level of IgG1 was only increased in the vaccinated mice at 70 dpv, compared with nonvaccinated mice (Figure 1). These results suggest that vaccination with Pru:Δcdpk2 in mice elicits a Th1-type immune response at 28 dpv and a mixed Th1/Th2 immune response at 70 dpv. Figure 1. View largeDownload slide Humoral response and antibody isotype profile in the serum of mice vaccinated with Pru:Δcdpk2 by intraperitoneal route. Levels of immunoglobulin G (IgG) and IgG subclass (IgG2a and IgG1) antibodies were evaluated in the sera of mice at 28 and 70 days after Pru:Δcdpk2 vaccination compared to nonvaccinated mice. The patterns of IgG2a to IgG1 show the induction of a type 1 T-helper (Th1) immune response at day 28 postvaccination, followed by a mixed Th1/type 2 T-helper immune response at day 70 postvaccination. Results are expressed as mean of optical density (OD) 450 mm ± standard deviation. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Figure 1. View largeDownload slide Humoral response and antibody isotype profile in the serum of mice vaccinated with Pru:Δcdpk2 by intraperitoneal route. Levels of immunoglobulin G (IgG) and IgG subclass (IgG2a and IgG1) antibodies were evaluated in the sera of mice at 28 and 70 days after Pru:Δcdpk2 vaccination compared to nonvaccinated mice. The patterns of IgG2a to IgG1 show the induction of a type 1 T-helper (Th1) immune response at day 28 postvaccination, followed by a mixed Th1/type 2 T-helper immune response at day 70 postvaccination. Results are expressed as mean of optical density (OD) 450 mm ± standard deviation. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Cytokine Production After Vaccination Cytokine production in splenocyte culture supernatants stimulated with STAg was assessed by ELISA at 70 dpv. The levels of Th1-type cytokines (IFN-γ, IL-2, and IL-12) were significantly higher than those in nonvaccinated mice. Also, the level of Th2-type cytokine (IL-10) was significantly higher than that of nonvaccinated mice (Figure 2). Figure 2. View largeDownload slide Levels of type 1 T-helper (Th1) and type 2 T-helper (Th2) cytokines produced by splenocytes culture of Pru:Δcdpk2-vaccinated mice. Spleen cells from 6 mice were obtained 70 days postvaccination and stimulated in vitro with 10 µg/mL soluble Toxoplasma gondii tachyzoite antigen. Cell-free supernatants were harvested and evaluated for Th1 (interferon gamma [IFN-γ], interleukin 2 [IL-2], and interleukin 12 [IL-12]) and Th2 (interleukin 10 [IL-10]) cytokines using enzyme-linked immunosorbent assay. Significance compared with control (uninfected + nonvaccinated) mice: **P < .01, ***P < .001. Figure 2. View largeDownload slide Levels of type 1 T-helper (Th1) and type 2 T-helper (Th2) cytokines produced by splenocytes culture of Pru:Δcdpk2-vaccinated mice. Spleen cells from 6 mice were obtained 70 days postvaccination and stimulated in vitro with 10 µg/mL soluble Toxoplasma gondii tachyzoite antigen. Cell-free supernatants were harvested and evaluated for Th1 (interferon gamma [IFN-γ], interleukin 2 [IL-2], and interleukin 12 [IL-12]) and Th2 (interleukin 10 [IL-10]) cytokines using enzyme-linked immunosorbent assay. Significance compared with control (uninfected + nonvaccinated) mice: **P < .01, ***P < .001. Pru:Δcdpk2 Immunogenicity and Potency for Protection Against Acute Infection Kunming mice were vaccinated with 500 Pru:Δcdpk2 tachyzoites and, 70 days later, challenged with 1000 tachyzoites of T. gondii type I RH strain, or ToxoDB#9 (PYS or TgC7) strain. As expected, all nonvaccinated mice infected with 1000 RH strain or ToxoDB#9 (PYS or TgC7) strains died within 10 days after infection, whereas all mice vaccinated survived regardless of the challenging strains (Figure 3). We further analyzed Th1 cytokines (IL-12 and IFN-γ) in the serum and peritoneal fluids at 7 days after infection. Significantly elevated levels of IL-12 and IFN-γ were found in the nonvaccinated + RH-infected mice, whereas only modestly elevated levels of the same cytokines was observed in Pru:Δcdpk2 vaccinated + RH-infected mice (Figure 4). Figure 3. View largeDownload slide Protection of mice against acute Toxoplasma gondii infection. Survival curves of Pru:Δcdpk2-vaccinated mice, challenged intraperitoneally with 103 tachyzoites of RH, PYS, or TgC7 strains 70 days postvaccination. The survival of mice was monitored for 35 days. A log-rank (Mantel–Cox) test showed significant difference in the survival rates between vaccinated + infected groups compared to nonvaccinated + infected groups (P = .003). All mice in the vaccinated groups remained alive at day 35 after infection, but all mice in the nonvaccinated groups died between day 7 and 10 after infection. In addition to the separate curves that show the difference in the survival rate between vaccinated and nonvaccinated mouse groups infected with RH, PYS, or TgC7 strains (A–C), the overall survival rates in all vaccinated mouse groups compared with the nonvaccinated mouse groups were combined in 1 graph (D). Figure 3. View largeDownload slide Protection of mice against acute Toxoplasma gondii infection. Survival curves of Pru:Δcdpk2-vaccinated mice, challenged intraperitoneally with 103 tachyzoites of RH, PYS, or TgC7 strains 70 days postvaccination. The survival of mice was monitored for 35 days. A log-rank (Mantel–Cox) test showed significant difference in the survival rates between vaccinated + infected groups compared to nonvaccinated + infected groups (P = .003). All mice in the vaccinated groups remained alive at day 35 after infection, but all mice in the nonvaccinated groups died between day 7 and 10 after infection. In addition to the separate curves that show the difference in the survival rate between vaccinated and nonvaccinated mouse groups infected with RH, PYS, or TgC7 strains (A–C), the overall survival rates in all vaccinated mouse groups compared with the nonvaccinated mouse groups were combined in 1 graph (D). Figure 4. View largeDownload slide Proinflammatory cytokines produced by Pru:Δcdpk2-vaccinated mice after infection with Toxoplasma gondii RH. Mice were challenged with 103 RH tachyzoites 70 days postvaccination and levels of interferon gamma (IFN-γ) and interleukin 12 (IL-12) in the serum and peritoneal washes were assessed by enzyme-linked immunosorbent assay at 7 days postinfection. Highest levels of proinflammatory cytokines were found in the nonvaccinated infected mice. Significance compared with uninfected and nonvaccinated control mice: **P < .01, ***P < .001. Figure 4. View largeDownload slide Proinflammatory cytokines produced by Pru:Δcdpk2-vaccinated mice after infection with Toxoplasma gondii RH. Mice were challenged with 103 RH tachyzoites 70 days postvaccination and levels of interferon gamma (IFN-γ) and interleukin 12 (IL-12) in the serum and peritoneal washes were assessed by enzyme-linked immunosorbent assay at 7 days postinfection. Highest levels of proinflammatory cytokines were found in the nonvaccinated infected mice. Significance compared with uninfected and nonvaccinated control mice: **P < .01, ***P < .001. Protection Against Chronic Infection The protective efficacy of vaccination with Pru:Δcdpk2 against chronic infection was investigated in Kunming mice at 70 dpv. All Pru:Δcdpk2-vaccinated mice survived infection with 20 T. gondii Pru parasite cysts, whereas only 40% of nonvaccinated infected mice survived (Figure 5A). At 35 days postchallenge, parasite cyst burden in the brain of surviving Pru:Δcdpk2-vaccinated compared with nonvaccinated mice was determined. Nonvaccinated mice challenged with 20 Pru cysts had 4296 ± 687 cysts per brain, whereas Pru:Δcdpk2-vaccinated mice challenged with the same number of Pru cysts had significantly fewer cysts per brain (78 ± 48 cysts/brain) (P < .001; Figure 5B). Figure 5. View largeDownload slide Vaccination with Pru:Δcdpk2 promoted survival and reduced brain cyst’s burden after infection. A, Survival curves following challenge of Pru:Δcdpk2-vaccinated mice with 20 cysts of Toxoplasma gondii Pru strain, compared with nonvaccinated mice, 70 days postvaccination. The survival of mice was monitored for 35 days. A log-rank (Mantel–Cox) test demonstrated a significant difference between the vaccinated and nonvaccinated groups (P = .0001). B, Cyst burden in the brain of mice that survived to 35 days after challenge (Pru:Δcdpk2-vaccinated + Pru-infected mice vs nonvaccinated + Pru-infected mice). Data points indicate mean ± SD. ***P < .001. Figure 5. View largeDownload slide Vaccination with Pru:Δcdpk2 promoted survival and reduced brain cyst’s burden after infection. A, Survival curves following challenge of Pru:Δcdpk2-vaccinated mice with 20 cysts of Toxoplasma gondii Pru strain, compared with nonvaccinated mice, 70 days postvaccination. The survival of mice was monitored for 35 days. A log-rank (Mantel–Cox) test demonstrated a significant difference between the vaccinated and nonvaccinated groups (P = .0001). B, Cyst burden in the brain of mice that survived to 35 days after challenge (Pru:Δcdpk2-vaccinated + Pru-infected mice vs nonvaccinated + Pru-infected mice). Data points indicate mean ± SD. ***P < .001. Protection Against Congenital Toxoplasmosis Pregnant mice were orally challenged with 10 T. gondii Pru cysts on day 12 of gestation, and the litter size and body weight of the neonates were determined. The cyst burden in the brain of neonates and dams was evaluated. The litter size and survival of pups of Pru:Δcdpk2-vaccinated + Pru cyst–infected dams was similar to that of nonvaccinated uninfected mice (Figure 6A). Body weight of neonates born to Pru:Δcdpk2-vaccinated + Pru cyst–infected dams was similar to neonates born to nonvaccinated uninfected dams (Figure 6B). In contrast, the litter size and body weight of pups of nonvaccinated + Pru cyst–infected mice were significantly lower compared with those of nonvaccinated uninfected mice or Pru:Δcdpk2-vaccinated + Pru cyst–infected mice. The brain cyst burden in the neonates was determined at day 35 postpartum. The average brain cyst number in all neonates (n = 19) born to nonvaccinated + Pru cyst–infected dams was 919 ± 339. In contrast, 41.4% (24/58) of neonates born to Pru:Δcdpk2-vaccinated + Pru cyst–infected dams had an average brain cyst number of (60 ± 33). Interestingly, examination of the brain of 58.6% (34/58) of neonates born to vaccinated + infected dams revealed no cysts. The brain cyst burden in dams was also determined at day 30 postpartum. The average brain cyst number was significantly higher in nonvaccinated + Pru cyst–infected dams (3287 ± 569 cysts/brain) than in that of Pru:Δcdpk2-vaccinated + Pru cyst–infected dams (77 ± 58 cysts/ brain). Figure 6. View largeDownload slide Protection of mice against type II Pru cyst infection on day 12 of gestation. A, Litter size and survival of pups from nonvaccinated uninfected mice, nonvaccinated + Pru-infected mice, and Pru:Δcdpk2-vaccinated + Pru-infected mice was assessed at birth and 35 days after birth. B, The average body weight of 35-day-old pups. Data points indicate mean ± standard deviation. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Figure 6. View largeDownload slide Protection of mice against type II Pru cyst infection on day 12 of gestation. A, Litter size and survival of pups from nonvaccinated uninfected mice, nonvaccinated + Pru-infected mice, and Pru:Δcdpk2-vaccinated + Pru-infected mice was assessed at birth and 35 days after birth. B, The average body weight of 35-day-old pups. Data points indicate mean ± standard deviation. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Immune Responses to Infection During Pregnancy The levels of T. gondii–specific Th1 (IFN-γ, IL-12, IL-2) and Th2 (IL-10) cytokines from Pru:Δcdpk2-vaccinated + Pru cyst–infected mice were significantly higher compared with those in nonvaccinated + Pru cyst–infected mice and nonvaccinated uninfected mice (P < .001; Figure 7). However, the difference in the cytokine levels between nonvaccinated + Pru cyst–infected and nonvaccinated uninfected mice was not statistically significant (P > .05). Figure 7. View largeDownload slide Profile of Toxoplasma gondii–specific type 1 T-helper (Th1) and type 2 T-helper (Th2) cytokines in Pru:Δcdpk2-vaccinated mice after infection with Pru cysts. Vaccinated, pregnant mice were infected with T. gondii Pru cysts at 12 days of gestation and spleen cells were collected 6 days after infection. Splenocyte culture was stimulated with 10 µg/mL T. gondii soluble tachyzoite antigen. Cell-free supernatants were evaluated for Th1 (interferon gamma [IFN-γ], interleukin 2 [IL-2], and interleukin 12 [IL-12]) and Th2 (interleukin 10 [IL-10]). Cytokine concentrations represent mean ± standard deviation after subtraction of background control values with medium only. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. Figure 7. View largeDownload slide Profile of Toxoplasma gondii–specific type 1 T-helper (Th1) and type 2 T-helper (Th2) cytokines in Pru:Δcdpk2-vaccinated mice after infection with Pru cysts. Vaccinated, pregnant mice were infected with T. gondii Pru cysts at 12 days of gestation and spleen cells were collected 6 days after infection. Splenocyte culture was stimulated with 10 µg/mL T. gondii soluble tachyzoite antigen. Cell-free supernatants were evaluated for Th1 (interferon gamma [IFN-γ], interleukin 2 [IL-2], and interleukin 12 [IL-12]) and Th2 (interleukin 10 [IL-10]). Cytokine concentrations represent mean ± standard deviation after subtraction of background control values with medium only. Significance compared with control (uninfected + nonvaccinated) mice: ***P < .001. DISCUSSION A highly effective vaccine against toxoplasmosis is urgently required, with leading vaccination strategies targeting both horizontal transmission and vertical transmission from pregnant dams to their offspring. Previous study has shown that cdpk2 is essential for the development of viable T. gondii tissue cysts, as cdpk2-deficient parasite strains failed to form cysts in the brain of mice [23]. Based on this observation, we inferred that cdpk2-deficient T. gondii might be a promising live attenuated vaccine to prevent toxoplasmosis. In the present study, cdpk2 was successfully disrupted in T. gondii RH and Pru strains using CRISPR-Cas9 method; the mutant strains exhibited the anticipated morphological abnormalities, and no parasite cyst was detected in the brain of mice challenged with Pru:Δcdpk2 tachyzoites, which was consistent with previous study [23]. Our results showed that deletion of the cdpk2 gene did not result in the attenuation of the virulence of RH or Pru strains, as indicated by the comparable survival rates of mice infected with the parental WT (RH or Pru) or mutant (RH:Δcdpk2 or Pru:Δcdpk2) strains. A high dose (105) of Pru:Δcdpk2 tachyzoites caused mice death in 11 days; however, all mice infected with the same strain, but with a smaller dose (500), survived and no parasite cyst was detected in their brain. Therefore, 500 Pru:Δcdpk2 tachyzoites were used in all subsequent experiments as the vaccination dose because it exhibited the type of balance between safety and high immunogenicity that should be expected of a promising live attenuated vaccine strain. Consistent with other attenuated T. gondii vaccines, vaccination with Pru:Δcdpk2 induced a high level of anti–T. gondii IgG antibodies in mice [12–18]. These specific IgG antibodies can neutralize the attachment of T. gondii and limit its ability to establish infection [35–38]. Pru:Δcdpk2-vaccinated mice also developed a sequential Th1 and a mixed Th1/Th2-type immune response, as indicated by the high level of Th1 immunity (IgG2a) at day 28 postvaccination; and the elevation of Th2 protective (IgG1) level at day 70 dpv when compared to that in nonvaccinated mice. The high level of IgG2a and IgG1 in Pru:Δcdpk2-vaccinated mice was substantiated by high levels of Th1 cytokines (IFN-γ, IL-2, and IL-12) and Th2 cytokines (IL-10) at 70 dpv. Indeed, the immune response induced by Pru:Δcdpk2 vaccination completely protected mice (ie, 100% survival rate) postchallenge with RH, or ToxoDB#9 (PYS or TgC7) strain, whereas nonvaccinated mice died within 10 dpi. In addition, Pru:Δcdpk2 significantly reduced the development of chronic toxoplasmosis. The parasite cysts were significantly reduced in the brain of vaccinated mice after infection with 20 cysts of T. gondii Pru strain compared with nonvaccinated + Pru cyst–infected mice. In our study, Th1 cytokines from the nonvaccinated + RH-infected mice were significantly higher than those from the Pru:Δcdpk2 vaccinated + RH-infected mice, indicating that vaccination with Pru:Δcdpk2 may have down-regulated Th1 response and reduced the severe inflammatory response that accompanies acute T. gondii infection [39–42]. Th1-type cytokine responses with high levels of IFN-γ, IL-2, and IL-12 are needed to limit congenital transmission and to protect pregnant mice against T. gondii infection [43–46]. However, Th1-type immune response can be detrimental to pregnancy and potentially deleterious for the conceptus [43, 44]. Thus, a balanced Th1/Th2 immune response detected at day 70 postvaccination seems to contribute to the observed successful pregnancy outcomes, as unsuccessful pregnancies were observed at day 28 postvaccination (data not shown) where Th1 response was dominating (Figure 1). Therefore, the high level of the Th2-related anti-inflammatory cytokine IL-10, which neutralizes the production of Th1 cytokines (IFN-γ and IL-12) detected in Pru:Δcdpk2-vaccinated pregnant mice infected with Pru cysts on day 12 of gestation (Figure 7) may have contributed to better pregnancy outcomes compared with nonvaccinated + Pru-infected pregnant mice. Higher IL-10 levels appear to play a role in protection against infection, because IL-10–deficient mice died during acute T. gondii infection due to a strong Th1 inflammatory response [47]. Previous studies showed that pregnancy outcomes can be improved in T. gondii–infected mice by administration of recombinant IL-10 and can be worsened in IL-10–deficient mice [48]. Despite the inhibitory effect of maternal and fetal Th2 polarized immune response that occurs during pregnancy on Th1 cytokine production, vaccination seems to elicit a protective immune response that enabled the pregnant mice to control the infection. These results show that vaccination of mice with Pru:Δcdpk2 achieved a critical balance between Th1 and Th2 responses for optimal control of infection, while minimizing overt inflammation and severe pathology. Although the immune responses induced by Pru:Δcdpk2 vaccination cannot completely block the vertical transmission, a statistically lower brain cyst burden was observed in pups from Pru:Δcdpk2-vaccinated + Pru-infected dams, compared to pups of nonvaccinated + Pru-infected dams. In addition, there were significantly fewer numbers of brain cysts in Pru:Δcdpk2-vaccinated + Pru-infected dams than in nonvaccinated + Pru-infected dams. Also, neonates born to Pru:Δcdpk2-vaccinated + Pru-infected dams had similar body weights to neonates born to nonvaccinated + uninfected mice. These results indicate that vaccination with Pru:Δcdpk2 can confer significant protection against subsequent oral infection with Pru cysts during the second trimester of gestation. Unexpectedly, infection with T. gondii Pru strain at 12 days of gestation failed to elicit more cytokines in nonvaccinated + Pru-infected mice, compared with nonvaccinated + uninfected mice (Figure 7). At present, the reason for this observation is unknown. However, it is possible that the time elapsed after infection was not long enough to elicit a detectable level of cytokines nor to induce potent CD8+ T-cell responses without prior antigenic stimulation. In conclusion, data reported in this study demonstrate that a single ip vaccination of 500 Pru:Δcdpk2 tachyzoites provides cross-protection against homologous and heterologous challenge with multiple T. gondii strains of the same and different genotypes in mice. Pru:Δcdpk2 strain generated an immune response, which improved the survival rate and reduced parasite cyst burden in the brain of the vaccinated mice. Strong protection against congenital toxoplasmosis was demonstrated by a significant reduction in the brain tissue cyst burden in the pups and their dams, along with improvement in the body weight and survival rate of pups born to vaccinated dams compared with nonvaccinated dams. Given the potential efficacy of Pru:Δcdpk2 live attenuated vaccine, the data generated in Kunming mice merit further exploration and should be evaluated in larger animals, such as sheep. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. The authors thank Luke Matthews for contributing to the design of the graphs and Paul Goodwin for critical comments on the manuscript. Author contributions. X. Q. Z., S. Y. H., and H. M. E. conceived the project, designed the experiments, and critically revised the manuscript. J. L. W., T. T. L., and K. C. performed the experiments and analyzed the data. J. L. W. drafted the manuscript. W. C., W. B. Y., and M. J. 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The Journal of Infectious DiseasesOxford University Press

Published: Apr 13, 2018

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